Saikosaponin D functionalized lipid nanoparticles, a preparation method thereof and application thereof in preparation of an antitumor drug

Abstract

The application discloses a saikosaponin D functionalized lipid nanoparticle, a preparation method thereof and application of the saikosaponin D functionalized lipid nanoparticle in preparation of an antitumor drug, and belongs to the technical field of biological medicine and nanomedicine. In the application, saikosaponin D is used as a membrane component of the lipid nanoparticle; the C-3 disaccharide structure of the saikosaponin D can be used as a molecular recognition motif of GLUT1, and the ability of the nanoparticle to actively target tumor cells is given; the pentacyclic triterpenoid skeleton of the saikosaponin D can be used as a membrane stabilizer instead of cholesterol, and meanwhile, lysosome retention mediated by NPC1 is avoided. The application also combines a photosensitizer, which can be selectively anchored in mitochondria by means of cation lipophilicity, and has the performance of precise photodynamic damage at the level of organelles; the obtained nanoparticle can significantly inhibit tumor growth of HCT-116 colorectal cancer tumor-bearing mice. The application integrates active targeting and efficient photodynamic therapy in a single preparation, and provides a safe and effective nanoplatform for tumor treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine, and in particular to a saikosaponin D-functionalized lipid nanoparticle, its preparation method, and its application in the preparation of antitumor drugs. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide, and its high recurrence rate and chemotherapy resistance remain major challenges in clinical treatment. Currently, clinical treatments for colorectal cancer mainly include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, with chemotherapy remaining the core treatment for advanced and metastatic colorectal cancer. However, traditional chemotherapy drugs (such as 5-fluorouracil, oxaliplatin, and irinotecan) suffer from poor targeting, low bioavailability, and strong toxic side effects (such as gastrointestinal reactions, bone marrow suppression, and neurotoxicity), severely impacting patient treatment adherence and quality of life.

[0003] Photodynamic therapy (PDT), a non-invasive treatment method, kills tumor cells by generating reactive oxygen species (ROS) through photosensitizers under specific wavelengths of light, and has received widespread attention in recent years. Near-infrared photosensitizer IR780 has advantages such as strong near-infrared absorption, high photostability, and mitochondrial attraction; however, free IR780 has poor water solubility, lacks tumor specificity, and is rapidly cleared from the body, severely limiting its clinical application.

[0004] Lipid nanoparticles (LNPs) are an important platform for photosensitizer encapsulation. Traditional LNPs use cholesterol as a membrane stabilizer, offering advantages such as high drug loading capacity, good biocompatibility, and scalable manufacturing processes. However, cholesterol-based LNPs have two drawbacks: first, they lack active targeting ligands, and tumor accumulation mainly relies on the enhanced permeation and retention (EPR) effect, which is often heterogeneous and inefficient in poorly vascularized tumor regions; second, cholesterol-containing LNPs are easily captured and degraded by lysosomes after entering cells, resulting in low photosensitizer bioavailability. These inherent drawbacks urgently require the development of alternative membrane components that can endow active tumor targeting capabilities and promote efficient subcellular delivery.

[0005] Saikosaponin D (SSD) is a pentacyclic triterpenoid saponin extracted from plants of the genus *Bupleurum*, with the chemical formula C0. 42 H 68 O 13With a molecular weight of 780.98, it possesses pharmacological effects such as antipyretic, sedative, anti-inflammatory, antibacterial, hepatoprotective, anti-nephritis, and immunomodulatory properties. Existing technologies have reported studies on the use of saikosaponin D for antidepressant, anti-epileptic, anti-inflammatory, and hepatoprotective purposes. However, there are currently no reports on using saikosaponin D as a membrane component of lipid nanoparticles (replacing cholesterol) and utilizing its glycosyl structure to achieve active targeting functions. Summary of the Invention

[0006] The purpose of this invention is to provide a saikosaponin D-functionalized lipid nanoparticle, its preparation method, and its application in the preparation of antitumor drugs.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention provides a saikosaponin D functionalized lipid nanoparticle, wherein the saikosaponin D functionalized lipid nanoparticle comprises a lipid bilayer and a photosensitizer encapsulated in the lipid bilayer; the lipid bilayer is composed of phospholipids, saikosaponin D and polyethylene glycol-modified phospholipids.

[0008] The second technical solution of this invention provides a method for preparing the above-mentioned saikosaponin D functionalized lipid nanoparticles, comprising the following steps: (1) Dissolve phospholipids, saikosaponin D and polyethylene glycol-modified phospholipids in an organic solvent and mix them evenly to obtain a mixed solution; (2) The photosensitizer is dissolved in dimethyl sulfoxide and then added to the mixed solution obtained in step (1). The organic solvent is then removed by rotary evaporation to obtain a drug-loaded lipid film. (3) Add ultrapure water to the drug-loaded lipid film obtained in step (2) for hydration, then perform probe ultrasonic treatment, and cool to room temperature to obtain the saikosaponin D functionalized lipid nanoparticles.

[0009] The third technical solution of this invention provides the application of the above-mentioned saikosaponin D functionalized lipid nanoparticles in the preparation of antitumor drugs.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention uses saikosaponin D as a membrane component of lipid nanoparticles to replace cholesterol. The C-3 disaccharide structure of saikosaponin D serves as a molecular recognition motif for glucose transporter 1 (GLUT1), endowing the nanoparticles with the ability to actively target tumor cells. The pentacyclic triterpenoid skeleton of saikosaponin D can replace cholesterol as a membrane stabilizer, while avoiding NPC1-mediated lysosomal retention. Furthermore, the epoxy ether bond in the aglycone of saikosaponin D is the key structural basis for the tumor microenvironment-responsive release of this invention—protonation and ring-opening under acidic conditions (pH=6.5) induces lipid bilayer instability and content release. In addition, the C-3 disaccharide structure of saikosaponin D provides a larger molecular recognition surface area and stronger GLUT1 binding affinity. This invention also incorporates a photosensitizer, which can selectively anchor to mitochondria through cationic lipophilicity, achieving precise photodynamic damage at the organelle level. The resulting nanoparticles significantly inhibit tumor growth in HCT-116 colorectal cancer-bearing mice. This invention integrates active targeting and highly efficient photodynamic therapy into a single formulation, providing a safe and effective nanoplatform for tumor treatment.

[0011] The saikosaponin D functionalized lipid nanoparticles provided by this invention have the following advantages: (1) Active targeting: This invention utilizes the C-3 disaccharide structure of saikosaponin D to specifically recognize GLUT1, which is highly expressed on the surface of tumor cells, and mediates the active targeting internalization of nanoparticles. The cell uptake rate decreased by 63.83% after pretreatment with the GLUT1 inhibitor Phloretin, confirming the GLUT1-mediated uptake mechanism. (2) Precise mitochondrial localization: The photosensitizer selectively anchors to mitochondria through cationic lipophilicity, with a colocalization coefficient of 0.94, achieving precise photodynamic damage at the organelle level. (3) pH-responsive release: The epoxy ether bond in the aglycone of saikosaponin D is protonated and ring-opened in the acidic environment of the tumor, inducing lipid bilayer instability and photosensitizer release, enhancing tumor-specific drug release. (4) Highly efficient photodynamic performance: Under 808 nm laser irradiation, the nanoparticles simultaneously drive a mixed photochemical pathway of type I (superoxide anion) and type II (singlet oxygen). At a concentration of 200 μg / mL, the total ROS generation efficiency can reach 76.08%, and the superoxide anion generation efficiency can reach 70.09%. Attached Figure Description

[0012] Figure 1 The particle size, polydispersity index (PDI), and zeta potential of the blank lipid nanoparticles of saikosaponin D obtained in Example 1 are shown in Figure 1, where A is the particle size, B is the polydispersity index (PDI), and C is the zeta potential. Figure 2The image shows the characterization of SLNPs@780 prepared in Example 2. In the image, A represents the particle size distribution and polydispersity index (PDI) of SLNPs@780, B represents the TEM image of SLNPs@780, C represents the Zeta potentials of SLNPs@780 and IR780, D represents the standard curve of IR780, E represents the encapsulation efficiency and drug loading of SLNPs@780, F represents the colloidal stability of SLNPs@780 stored at 4°C for 5 days, and G represents the particle size response of SLNPs@780 under simulated tumor microenvironment (pH=6.5). Figure 3 The image shows the photodynamic activity (PDT) evaluation of SLNPs@780 prepared in Example 2. In this image, A represents the change in absorbance with laser irradiation time during NBT detection, B represents the change in absorbance with SLNPs@780 concentration during NBT detection, and C represents the change in O2· during NBT detection. - The relationship between content and SLNPs@780 concentration, where D represents the change in absorbance value with laser irradiation time during DPBF detection, E represents the change in absorbance value with SLNPs@780 concentration during DPBF detection, and F represents the change in absorbance and concentration during DPBF detection. 1 The relationship between O2 content and SLNPs@780 concentration; Figure 4 The image shows the in vitro tumor cell uptake assessment of SLNPs@780 prepared in Example 2. In this image, A is the CLSM image of mitochondrial colocalization of SLNPs@780, B is the Pearson colocalization coefficient of SLNPs@780, C is the flow cytometry histogram of SLNPs@780, D is the flow cytometry mean fluorescence intensity quantification of SLNPs@780, E is the CLSM image of SLNPs@780 after Phloretin pretreatment, and F is the fluorescence intensity quantification analysis of SLNPs@780 after Phloretin pretreatment. Figure 5 The image shows the in vitro antitumor effect of SLNPs@780 prepared in Example 2. In the image, A is the CLSM image of SLNPs@780 mitochondrial membrane potential detected by the JC-1 probe; B is the quantitative quantification of the JC-1 red / green fluorescence ratio of SLNPs@780 without laser irradiation; C is the quantitative quantification of the JC-1 red / green fluorescence ratio of SLNPs@780 under laser irradiation; D is the CLSM image of SLNPs@780 stained with Calcein-AM / PI live / dead cells; E is the quantitative quantification of the percentage of live / dead cells in SLNPs@780 without laser irradiation; and F is the quantitative quantification of the percentage of live / dead cells in SLNPs@780 under laser irradiation. Figure 6The in vivo biodistribution map of SLNPs@780 prepared in Example 2 is shown in the following figures: A is the IVIS in vivo imaging dynamic map of SLNPs@780, B is the fluorescence intensity of SLNPs@780 at different time points, C is the in vitro organ fluorescence imaging of SLNPs@780, and D is the in vitro fluorescence intensity of SLNPs@780. Figure 7 The in vivo antitumor effect of SLNPs@780 prepared in Example 2 is shown in Figure A, where A is the actual image of mouse tumors in different treatment groups and the tumor growth curves of all mice, B is an overview of the tumor growth curves in different treatment groups, C is the actual image of ex vivo tumors in different treatment groups, D is the tumor weight in different treatment groups, E is the body weight change curve in different treatment groups, and F is the H&E staining results in different treatment groups. Detailed Implementation

[0013] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0014] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0015] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0016] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0017] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0018] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0019] This invention provides saikosaponin D-functionalized lipid nanoparticles, which comprise a lipid bilayer and a photosensitizer encapsulated within the lipid bilayer. The lipid bilayer is composed of phospholipids, saikosaponin D, and polyethylene glycol-modified phospholipids. This invention uses saikosaponin D instead of cholesterol as a membrane stabilizer, endowing the nanoparticles with the ability to actively target tumor cells while avoiding NPC1-mediated lysosomal retention. Combined with a photosensitizer, the nanoparticles can selectively anchor to mitochondria through cationic lipophilicity, achieving precise photodynamic damage at the organelle level. The resulting nanoparticles significantly inhibit tumor growth in HCT-116 colorectal cancer-bearing mice. This invention integrates active targeting and highly efficient photodynamic therapy into a single formulation, providing a safe and effective nanoplatform for tumor treatment.

[0020] In this invention, the mass ratio of phospholipids, saikosaponin D, and polyethylene glycol-modified phospholipids is 8:1:1. Controlling the mass ratio of phospholipids, saikosaponin D, and polyethylene glycol-modified phospholipids within the above range is beneficial for obtaining lipid nanocarriers with suitable particle size, good uniformity, and stable charge.

[0021] In this invention, the phospholipid is selected from soybean phosphatidylcholine; the polyethylene glycol-modified phospholipid is selected from DSPE-PEG. 2000 .

[0022] In this invention, the mass of the photosensitizer is 2% of the mass of the lipid bilayer.

[0023] In this invention, the photosensitizer encapsulation rate in the saikosaponin D functionalized lipid nanoparticles is ≥90%, and the drug loading of the photosensitizer is 6.1%.

[0024] In this invention, the photosensitizer is selected from IR780. IR780 has an excitation wavelength of 808 nm, which falls within the near-infrared window. Compared to traditional visible light photosensitizers (such as Photofrin, with an excitation wavelength of 630 nm), near-infrared light has a significantly greater penetration depth in biological tissues (up to several centimeters), making it suitable for the treatment of deep tumors. IR780 also exhibits cationic lipophilicity, allowing it to spontaneously anchor to mitochondria via the mitochondrial membrane potential (approximately -180 mV), achieving precise localization at the organelle level. This enables it to target GLUT1 of saikosaponin D in a stepwise, precise delivery process, targeting cells first and then mitochondria. Furthermore, IR780 itself possesses near-infrared fluorescence properties, which can be used for in vivo fluorescence imaging, enabling real-time monitoring during treatment. IR780 also exhibits high photostability and is not prone to photobleaching.

[0025] In this invention, the saikosaponin D functionalized lipid nanoparticles have a particle size of 83.54±2.1 nm, a polydispersity index (PDI) of 0.19±0.05, and a zeta potential of -24.77±2.7 mV.

[0026] This invention also provides a method for preparing the above-mentioned saikosaponin D functionalized lipid nanoparticles, comprising the following steps: (1) Dissolve phospholipids, saikosaponin D and polyethylene glycol-modified phospholipids in an organic solvent and mix them evenly to obtain a mixed solution; (2) The photosensitizer is dissolved in dimethyl sulfoxide and then added to the mixed solution obtained in step (1). The organic solvent is then removed by rotary evaporation to obtain a drug-loaded lipid film. (3) Add ultrapure water to the drug-loaded lipid film obtained in step (2) for hydration, then perform probe ultrasonic treatment, and cool to room temperature to obtain the saikosaponin D functionalized lipid nanoparticles.

[0027] In this invention, in step (1), the organic solvent is selected from one or more of dichloromethane, methanol and anhydrous ethanol, and is more preferably dichloromethane, methanol and anhydrous ethanol; the volume ratio of dichloromethane, methanol and anhydrous ethanol is 2:1:1.

[0028] In this invention, in step (2), the temperature of the rotary evaporation is 40~50℃, more preferably 45℃; the vacuum degree of the rotary evaporation is -0.08~-0.1 MPa; and the rotation speed of the rotary evaporation is 1000 rpm.

[0029] In this invention, step (2) further includes a drying step after the rotary evaporation is completed; the drying temperature is room temperature, the drying time is 12 hours, and the drying equipment is a vacuum drying oven. This invention thoroughly removes residual organic solvents through drying.

[0030] In this invention, in step (3), the amount of ultrapure water used is 2 mL of ultrapure water added for every 10 mg of total lipids, wherein total lipids refer to the total amount of phospholipids, saikosaponin D and polyethylene glycol phospholipids; the hydration temperature is 40°C and the hydration time is 30 min; the hydration is carried out under magnetic stirring.

[0031] In this invention, in step (3), the power of the probe ultrasound treatment is 40~60 W, more preferably 50 W; the probe ultrasound treatment is performed intermittently, specifically 5 seconds of ultrasound followed by 2 seconds of pause, and the total time of the probe ultrasound treatment is 5~10 minutes; the device for the probe ultrasound treatment is a cell probe disruptor.

[0032] This invention also provides the application of the above-mentioned saikosaponin D functionalized lipid nanoparticles in the preparation of antitumor drugs.

[0033] In this invention, the tumor is colorectal cancer; the colorectal cancer cells are HCT-116 cells; and the antitumor drug is a photodynamic therapy drug activated by an 808 nm near-infrared laser.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] In the following examples, saikosaponin D was purchased from Beijing Herbalife Technology Co., Ltd. (CAS: 20874-52-6, catalog number CFN9989), soybean phosphatidylcholine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and DSPE-PEG was used. 2000 The cells were purchased from Chongqing Yusi Technology Co., Ltd., IR780 from Shanghai Bid Pharmaceutical Technology Co., Ltd., and HCT-116 cell line (catalog number FH0027) from Shanghai Fuheng Biotechnology Co., Ltd.

[0036] Example 1 A method for preparing blank lipid nanoparticles of saikosaponin D (Blank-SLNPs) includes the following steps: (1) Soybean phosphatidylcholine (PC), saikosaponin D (SSD) and DSPE-PEG were added. 2000 The components were dissolved in a mixed organic solvent of dichloromethane, methanol, and anhydrous ethanol at mass ratios of 5:1:1, 6:1:1, 7:1:1, 8:1:1, 9:1:1, 10:1:1, 8:5:1, 8:3:1, 8:1:5, and 8:1:3, respectively. The volume ratio of dichloromethane, methanol, and anhydrous ethanol was 2:1:1. The mixtures were stirred thoroughly to ensure complete dissolution of all components, resulting in a mixed solution.

[0037] (2) Transfer the mixed solution obtained in step (1) to a round-bottom flask, and then place it on a rotary evaporator. Set the water bath temperature to 45°C, the vacuum degree to -0.08~-0.1 MPa, and the rotation speed to 1000 rpm. Evaporate the organic solvent under reduced pressure until a uniform and transparent lipid film is formed on the inner wall of the flask. Remove the round-bottom flask from the rotary evaporator and place it in a vacuum drying oven to dry at room temperature overnight (12 h) to completely remove the residual organic solvent and obtain the lipid film.

[0038] (3) Add ultrapure water to the round-bottom flask containing the lipid film in step (2). The amount of ultrapure water is calculated at a ratio of 2 mL of ultrapure water per 10 mg of total lipids. Then, place the round-bottom flask in a 40°C water bath and hydrate it with magnetic stirring for 30 min to fully hydrate the lipid film and allow it to detach from the flask wall. Subsequently, use a cell probe disruptor (power 50W) to sonicate the probe, sonicating for 5 s with a 2 s pause, for a total sonication time of 5 min, until the dispersion is a uniform suspension with a translucent blue opalescent appearance. Allow it to cool naturally to room temperature to obtain blank lipid nanoparticles of saikosaponin D (Blank-SLNPs). Aliquot the obtained blank lipid nanoparticles into cryovials and store them at -20°C for later use.

[0039] Figure 1 The particle size, polydispersity index (PDI), and zeta potential of the blank lipid nanoparticles of saikosaponin D obtained in Example 1 are shown, where A is the particle size, B is the PDI, and C is the zeta potential. Particle size affects the in vivo circulation time and tumor tissue penetration efficiency of the nanoparticles; the polydispersity index (PDI) reflects the uniformity of particle size distribution, with PDI < 0.3 indicating a monodisperse state; the absolute value of the zeta potential affects the electrostatic repulsion between particles, thus affecting storage stability and aggregation tendency during in vivo circulation. The screening criteria were set as: average particle size < 100 nm, PDI < 0.2, and absolute zeta potential > 20 mV. Figure 1 By evaluating the particle size, polydispersity index (PDI), and zeta potential of the blank lipid nanoparticles prepared with saiko saponin D at ten different mass ratios in Example 1, soybean phosphatidylcholine, saiko saponin D, and DSPE-PEG were screened out. 2000 The blank lipid nanoparticles of saikosaponin D with a mass ratio of 8:1:1 are lipid nanocarriers with suitable particle size, good uniformity and stable charge.

[0040] Example 2 A method for preparing saikosaponin D-functionalized lipid nanoparticles (SLNPs@780), the specific steps of which are as follows: (1) Soybean phosphatidylcholine (PC), saikosaponin D (SSD) and DSPE-PEG were added. 2000 Dissolve the components in a mixed organic solvent of dichloromethane, methanol, and anhydrous ethanol at a mass ratio of 8:1:1 and a volume ratio of 2:1:1. Stir thoroughly to ensure complete dissolution of all components and obtain a mixed solution.

[0041] (2) The photosensitizer IR780 was dissolved in dimethyl sulfoxide (DMSO) to prepare an IR780 stock solution with a concentration of 1 mg / mL. The IR780 stock solution was added to the mixed solution obtained in step (1) at a ratio of 2% of the total lipid mass. The mixture was stirred thoroughly for 10 min to ensure that the IR780 and lipid components were evenly mixed. The mixture was then transferred to a round-bottom flask and placed on a rotary evaporator. The water bath temperature was set to 45°C, the vacuum degree was controlled at -0.08 to -0.1 MPa, and the rotation speed was set to 1000 rpm. The organic solvent was removed by vacuum evaporation. As the solvent evaporated, a uniform blue-green drug-loaded lipid film gradually formed on the inner wall of the flask. The round-bottom flask was removed from the rotary evaporator and placed in a vacuum drying oven to dry at room temperature overnight (12 h) to completely remove the residual organic solvent and obtain the drug-loaded lipid film.

[0042] (3) Add ultrapure water to the round-bottom flask containing the drug-loaded lipid film in step (2). The amount of ultrapure water is calculated at a ratio of 2 mL of ultrapure water per 10 mg of total lipids. Then, place the round-bottom flask in a 40°C water bath and magnetically stir for 30 min to hydrate the drug-loaded lipid film, allowing it to fully hydrate and detach from the flask wall, resulting in a transparent blue-green aqueous dispersion. Subsequently, use a cell probe disruptor (power 50 W) to sonicate the obtained aqueous dispersion with a probe, sonicating for 5 s and pausing for 2 s, for a total sonication time of 5 min, until the dispersion becomes a translucent blue-green uniform suspension. Allow it to cool naturally to room temperature to obtain IR780-loaded saikosaponin D-functionalized lipid nanoparticles (SLNPs@780). Aliquot the obtained SLNPs@780 into cryovials and store them at -20°C for later use.

[0043] Performance testing: 1. Particle size, polydispersity index, zeta potential, morphology, encapsulation efficiency and drug loading, stability, and pH responsiveness of SLNPs@780 1.1 Particle size, polydispersity index, zeta potential and morphology of SLNPs@780 The particle size, polydispersity index (PDI), and zeta potential of the SLNPs@780 prepared in Example 2 were determined using a dynamic light scattering instrument (Malvern Zetasizer ZS-90, Malvern Instruments Ltd., UK). The results are shown in the figure. Figure 2 Parts A and C are shown in the figure. SLNPs@780 prepared in Example 2 were dropped onto a copper mesh carbon film. After natural drying, the morphology of the nanoparticles was observed using a transmission electron microscope (TEM, Hitachi HT7700, Japan) at an accelerating voltage of 120 kV. The results are shown in the figure. Figure 2 Part B of the document.

[0044] 1.2 Encapsulation efficiency and drug loading of SLNPs@780 The encapsulation efficiency and drug loading of IR780 in the SLNPs@780 prepared in Example 2 were determined using an ultrafiltration-centrifugation method. 1 mL of the SLNPs@780 prepared in Example 2 was added to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4000 rpm for 10 min at 4°C. This allowed free IR780 to pass through the ultrafiltration membrane into the filtrate, while the lipid nanoparticles were retained within the ultrafiltration tube. The filtrate was collected, and the fluorescence intensity of free IR780 in the filtrate was measured at an excitation wavelength of 780 nm using a UV spectrophotometer (F-2700, Hitachi, Japan). An equal volume of the SLNPs@780 prepared in Example 2 was taken, and methanol was added at a 1:1 volume ratio to disrupt the lipid bilayer, allowing complete release of the encapsulated IR780. The absorbance of the total IR780 was then measured. The absorbance was determined according to the IR780 standard curve (see...). Figure 2 The IR780 content in each component was calculated using Part D of the formula. Encapsulation efficiency (EE) and drug loading (DL) were calculated using the following formulas, and the results are shown in [reference needed]. Figure 2 Part E in: EE (%) = (W) total - W free ) / W total ×100%; DL (%) = W total / (W) carrier + W total ) × 100%; In the formula, W total For the mass of IR780 encapsulated by lipid nanoparticles, W free W represents the mass of the lower layer of free IR780 in the ultrafiltrate. carrier Total mass of lipid carriers (phospholipids, saikosaponin D and DSPE-PEG) 2000 (The sum of their masses).

[0045] 1.3 Stability of SLNPs@780 To demonstrate the stability of SLNPs@780, the particle size and polydispersity index (PDI) of SLNPs@780 prepared in Example 2 were determined using a dynamic light scattering instrument (Malvern Zetasizer ZS-90, Malvern Instruments Ltd., UK) after storage at 4°C for 5 days. The results are shown in [Figure number missing]. Figure 2 The F part in the text.

[0046] 1.4 pH responsiveness of SLNPs@780 PBS (pH 6.5) was prepared to simulate the acidic microenvironment of tumors, and the temperature was maintained at 37 ± 0.5 °C. Two mL of SLNPs@780 (IR780 concentration 50 μg / mL) prepared in Example 2 was co-cultured in PBS at pH 6.5 for 6 h. The particle size change of SLNPs@780 was measured using a dynamic light scattering instrument (Malvern Zetasizer ZS-90, Malvern Instruments Ltd., UK). The results are shown in [Figure number missing]. Figure 2 The G part in the text.

[0047] Figure 2 Characterization diagrams of SLNPs@780 prepared in Example 2 are shown below. A represents the particle size distribution and polydispersity index (PDI) of SLNPs@780; B is a TEM image of SLNPs@780; C represents the Zeta potentials of SLNPs@780 and IR780; D is the standard curve of IR780; E represents the encapsulation efficiency and drug loading of SLNPs@780; F represents the colloidal stability of SLNPs@780 stored at 4°C for 5 days; and G represents the particle size response of SLNPs@780 under simulated tumor microenvironment (pH=6.5). Figure 2 As can be seen from Part A, the average hydrated particle size of SLNPs@780 is 83.54±2.1 nm, and the polydispersity index (PDI) is 0.19±0.05. Figure 2 The TEM image in section B shows that the SLNPs@780 nanoparticles are regular spherical, with a round and uniform morphology, good size, and good dispersibility, consistent with the DLS measurement results. Figure 2 As can be seen from section C, the Zeta potential of SLNPs@780 is -24.77 ± 2.7 mV. From... Figure 2 As can be seen from part E, the encapsulation efficiency of SLNPs@780 is 91.5%, and the drug loading is 6.1%. From... Figure 2 As can be seen from the F section, the particle size and PDI of SLNPs@780 did not change significantly within 5 days. Figure 2 As can be seen from part G in PBS at pH 6.5, the particle size of SLNPs@780 changed from a single peak to multiple peaks, indicating that SLNPs@780 has a tumor microenvironment response.

[0048] 2. In vitro ROS generation capacity of SLNPs@780 To systematically characterize the photodynamic therapy performance of SLNPs@780, two sets of cell-free in vitro spectroscopic experiments were conducted to quantify the type I photodynamic product, superoxide anion (O2·). - ) and type II photodynamic product singlet oxygen ( 1 O2).

[0049] Superoxide anion (O2· -Detection (Type I pathway): Quantitative analysis of O2· generated by the type I electron transfer photodynamic reaction using a nitrotetrazole blue (NBT) probe. - The capability. SLNPs@780 via 808 nm laser (1 W / cm²) 2 After irradiation, a type I photodynamic reaction occurs to generate O2· - O2· - Water-soluble yellow NBT can be reduced to an insoluble blue formazan precipitate; O2· - The concentration was positively correlated with the absorbance value measured at 560 nm. NBT was mixed with SLNPs@780 (concentration 100 μg / mL) and then... (The sentence is incomplete and requires more context to translate accurately.) 2 The UV-Vis absorption spectra were recorded in real time under 808 nm laser gradient irradiation (1 min, 2 min, 3 min, 4 min, 5 min) for different irradiation durations. The results are shown in [Figure number missing]. Figure 3 Part A of the formula: NBT was mixed with different concentrations of SLNPs@780 (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively) and then injected at 1 W / cm². 2 Irradiation with an 808 nm laser for 5 min was performed, and the UV-Vis absorption spectra at different concentrations were recorded in real time. The results are shown in [Figure number missing]. Figure 3 Part B of the text; according to Figure 3 In part B, the absorbance value corresponding to 560nm is plotted for O2· - The relationship between the content and the concentration of SLNPs@780 is shown in the figure. Figure 3 Part C of the text.

[0050] Singlet oxygen ( 1 O2 detection (Type II pathway): The type II photodynamic response was evaluated using the singlet oxygen-specific capture probe 1,3-diphenylisobenzofuran (DPBF). 1 O2 generation capacity. After mixing DPBF with SLNPs@780 (concentration 100 μg / mL) (DPBF initial absorbance calibrated to approximately 1.2 at 410 nm), it was analyzed at 1 W / cm². 2 The UV-Vis absorption spectra were recorded in real time under 808 nm laser gradient irradiation (1 min, 2 min, 3 min, 4 min, 5 min) for different irradiation durations. The results are shown in [Figure number missing]. Figure 3 Part D of the formula; DPBF was mixed with different concentrations of SLNPs@780 (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively) and then injected at 1 W / cm 2Irradiation with an 808 nm laser for 5 min was performed, and the UV-Vis absorption spectra at different concentrations were recorded in real time. The results are shown in [Figure number missing]. Figure 3 Part E in; according to Figure 3 In the E part, the absorbance value corresponding to 410 nm is plotted as absorbance and 1 The relationship between O2 content and SLNPs@780 concentration is shown in the figure. Figure 3 The F part in the text.

[0051] Figure 3 The image shows the photodynamic activity (PDT) evaluation of SLNPs@780 prepared in Example 2. In this image, A represents the change in absorbance with laser irradiation time during NBT detection, B represents the change in absorbance with SLNPs@780 concentration during NBT detection, and C represents the change in O2· during NBT detection. - The relationship between content and SLNPs@780 concentration, where D represents the change in absorbance value with laser irradiation time during DPBF detection, E represents the change in absorbance value with SLNPs@780 concentration during DPBF detection, and F represents the change in absorbance and concentration during DPBF detection. 1 The relationship between O2 content and SLNPs@780 concentration. From Figure 3 As shown in sections A to C, the absorption intensity of the NBT reduction product formazan at 560 nm continuously increases with laser irradiation time and SLNPs@780 concentration, with a superoxide anion generation efficiency of 70.09% at a concentration of 200 μg / mL. From... Figure 3 As can be seen from the D~F section, the characteristic absorption peak of DPBF at 410 nm gradually decays with the duration of illumination, and the absorbance of DPBF in the 200 μg / mL concentration group drops to 90.51% of the initial value at the end of the illumination period. The above results confirm that SLNPs@780 simultaneously drive both type I and type II photochemical pathways under photoexcitation.

[0052] 3. In vitro cellular uptake of SLNPs@780 and the GLUT1-mediated mechanism HCT-116 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. HCT-116 cells in the logarithmic growth phase were then cultured at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well in 6-well plates and incubated overnight (12 h) at 37°C to allow cell adhesion. The following day, cells were co-incubated with IR780, Lip@780 (traditional lipid nanoparticles containing cholesterol), and SLNPs@780, respectively, at an equivalent concentration of 5 μg / mL for 4 h and 8 h. After incubation, the culture medium was discarded, and cells were washed three times with pre-cooled PBS to remove residual nanoparticles and free fluorescent dyes. Cell uptake was observed using flow cytometry; results are shown below. Figure 4 The C and D parts of the [material / structure]. The preparation process of Lip@780 (traditional lipid nanoparticles containing cholesterol) is as follows: [The process involves] PC, cholesterol, and DSPE-PEG. 2000 Accurately weigh PC, cholesterol, and DSPE-PEG in a mass ratio of 5:1:1. 2000 Weigh out IR780, which accounts for 2% of the total lipid mass. Dissolve all the above components in dichloromethane and stir until completely dissolved to obtain a mixed solution. Add the resulting mixed solution to a round-bottom flask and remove the organic solvent by rotary evaporation under reduced pressure at 45°C. Continue evaporation until a uniform, transparent blue-green lipid film forms on the inner wall of the flask. Then, add ultrapure water to the round-bottom flask containing the lipid film, using a ratio of 2 mL of ultrapure water per 10 mg of total lipids. Place the round-bottom flask in a 40°C water bath and magnetically stir for 30 min to hydrate the lipid film completely, allowing it to detach and disperse. Afterward, use a probe sonicator (50 W power) to sonicate (5 s sonication, 2 s pause, total duration 5 min) until a translucent, uniformly dispersed, blue-opalescent dispersion is obtained. Freeze-dry using a freeze dryer to obtain the freeze-dried powder Lip@780.

[0053] Mitochondrial colocalization assay: HCT-116 cells were seeded in confocal microarrays and co-incubated for 12 h with IR780, Lip@780, and SLNPs@780 (IR780 concentration 5 μg / mL for each cell). After incubation, cells were washed three times with PBS, and MitoTracker Green working solution (final concentration 200 nM) was added. The cells were then incubated at 37°C in the dark for 30 min to label mitochondria. After washing three times with PBS, cells were fixed with 4% paraformaldehyde for 20 min, and images were acquired using a CLSM scanner. The results are shown in the figure below. Figure 4 Part A of the diagram. Pearson colocalization coefficients were analyzed using ImageJ software; the results are shown in [Figure A]. Figure 4 Part B of the document.

[0054] GLUT1 inhibition assay: HCT-116 cells were seeded in confocal microplates and pretreated with the GLUT1-specific inhibitor Phloretin (20 μM) for 30 min after adhesion. Then, SLNPs@780 and Lip@780 (IR780 concentration 5 μg / mL) were added and incubated for another 4 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde, stained with DAPI, and images were acquired using CLSM. The results are shown below. Figure 4 The E~F parts in the text.

[0055] Figure 4 The image shows the in vitro tumor cell uptake assessment of SLNPs@780 prepared in Example 2. In this image, A is the CLSM image of SLNPs@780 mitochondrial colocalization, B is the Pearson colocalization coefficient of SLNPs@780, C is the flow cytometry histogram of SLNPs@780, D is the flow cytometry mean fluorescence intensity quantification of SLNPs@780, E is the CLSM image of SLNPs@780 after Phloretin pretreatment, and F is the fluorescence intensity quantification analysis of SLNPs@780 after Phloretin pretreatment. Figure 4 As can be seen in section A, the red fluorescence of SLNPs@780 highly overlaps with the green fluorescence of MitoTracker Green. From... Figure 4 As can be seen from Part B, the Pearson colocation coefficient of SLNPs@780 reaches 0.94, while the colocation degree of Lip@780 and IR780 groups is almost negligible. Figure 4 Flow cytometry analysis of the C-D segments showed that the average fluorescence intensity of SLNPs@780 at 4 h and 8 h was more than five times that of Lip@780. Figure 4 As shown in sections E-F, phloretin pretreatment significantly reduced cellular uptake of SLNPs@780, with an intracellular mean fluorescence intensity reduction of 63.83%; while Lip@780 uptake was almost unaffected by phloretin. This indicates that the internalization of SLNPs@780 mainly depends on the GLUT1-mediated active transport pathway.

[0056] 4. In vitro antitumor effects of SLNPs@780 Mitochondrial membrane potential detection: HCT-116 cells were prepared at a ratio of 1×10⁻⁶. 5 Individual inoculated cells were placed in confocal microplates and co-incubated with IR780, Lip@780, and SLNPs@780 for 4 h, respectively. Then, an 808 nm laser irradiation group (1 W / cm²) was established. 2The incubation period was 5 min, with (+L) and a non-irradiated control group (-L). After incubation, the cells were washed three times with PBS, and JC-1 working solution (final concentration 5 μg / mL) was added. The cells were incubated at 37°C in the dark for 20 min. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 20 min. Images were acquired using CLSM, and the results are shown in the table below. Figure 5 Part A of the diagram. JC-1 forms a red fluorescent polymer (aggregate state, emitting approximately 590 nm) in healthy mitochondria due to high membrane potential, while it exists as a green fluorescent monomer (emitting approximately 530 nm) when the membrane potential is lost. The red / green fluorescence ratio of JC-1 at SLNPs@780 was used for quantification, and the results are shown in [Figure 1]. Figure 5 Parts B to C in the text.

[0057] Live and dead cell staining: Cell viability was assessed using Calcein-AM / PI dual fluorescence staining. HCT-116 cells were co-incubated with IR780, Lip@780, and SLNPs@780 for 4 h, respectively. Cells were then divided into an 808 nm laser irradiation group (+L) and a non-irradiation control group (-L). After washing three times with PBS, Calcein-AM / PI mixed staining solution (calcein-AM final concentration 2 μM, PI final concentration 4.5 μM) was added, and the cells were incubated at 37°C in the dark for 30 min. Images were acquired using CLSM after PBS washing; results are shown below. Figure 5 Part D of the diagram. Live cells were labeled with Calcein-AM (green fluorescence), and dead cells were labeled with PI (red fluorescence). The percentage of live / dead cells at SLNPs@780 was quantified. Results are shown in [Figure number missing]. Figure 5 The E~F parts in the text.

[0058] Figure 5 The image shows the in vitro antitumor effect of SLNPs@780 prepared in Example 2. In the image, A is the CLSM image of SLNPs@780 mitochondrial membrane potential detected by the JC-1 probe; B is the quantitative quantification of the JC-1 red / green fluorescence ratio of SLNPs@780 without laser irradiation; C is the quantitative quantification of the JC-1 red / green fluorescence ratio of SLNPs@780 under laser irradiation; D is the CLSM image of SLNPs@780 stained with Calcein-AM / PI live / dead cells; E is the quantitative quantification of the percentage of live / dead cells in SLNPs@780 without laser irradiation; and F is the quantitative quantification of the percentage of live / dead cells in SLNPs@780 under laser irradiation. Figure 5 The results in sections A through C show that the red / green fluorescence ratios of each group were basically the same under the condition of no laser irradiation; however, after irradiation with an 808 nm laser, the red fluorescence of the SLNPs@780 group was significantly weakened and the green fluorescence was enhanced, with the red / green ratio decreasing by 3.65 times that of the IR780 group and 2.27 times that of the Lip@780 group. Figure 5The results in sections D to F showed that the SLNPs@780(+L) group exhibited large areas of red fluorescence, the green live cell signal almost disappeared, and the dead cell ratio reached 50.47%, which was 8.92 times that of the IR780 group and 2.20 times that of the Lip@780 group. Under the condition of no light, the live and dead cell ratios of each group were basically the same.

[0059] 5. In vivo biodistribution of SLNPs@780 Establishment of HCT-116 tumor-bearing mouse model: BALB / c nude mice (male, 4-6 weeks old, weighing 16-18g) were acclimatized for one week. HCT-116 cells in the logarithmic growth phase were digested with trypsin and prepared into 1×10⁻⁶ cells using pre-cooled PBS. 7 A cell suspension of 0.2 mL / mL was subcutaneously injected into the right axilla of nude mice. Tumors were allowed to grow to approximately 100 mm in size. 3 Experiments were conducted at that time.

[0060] Tumor-bearing mice were randomly divided into 3 groups ( n =3 / group): IR780 group, Lip@780 group, and SLNPs@780 group. The IR780 dose was 1 mg / kg, administered via tail vein injection. At predetermined time points (0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h), mice were subjected to fluorescence imaging using a small animal in vivo imaging system (IVIS Spectrum, PerkinElmer). The excitation wavelength was 745 nm, and the emission wavelength was 820 nm. Results are shown below. Figure 6 Part A of the data; and the fluorescence intensity at different time points was analyzed, the results are shown in [Figure A]. Figure 6 Part B of the study. After 24 hours of imaging, mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for in vitro fluorescence imaging. The results are shown in [Figure B]. Figure 6 Part C of the study; and quantitative analysis of the fluorescence intensity of each organ, the results are shown in [Figure number missing]. Figure 6 Part D in the text.

[0061] Figure 6 The image shows the in vivo biodistribution of SLNPs@780 prepared in Example 2. In this image, A is a dynamic IVIS in vivo imaging image of SLNPs@780, B is the fluorescence intensity of SLNPs@780 at different time points, C is the fluorescence imaging of SLNPs@780 in ex vivo organs, and D is the fluorescence intensity of SLNPs@780 in ex vivo. Figure 6 The results in sections A to B showed that specific fluorescence signals could be observed at the tumor site 1 hour after SLNPs@780 injection, and the fluorescence intensity reached its peak at 4 hours, which was significantly higher than that of the Lip@780 group and the IR780 group. The fluorescence signal could still be detected up to 24 hours. In contrast, the fluorescence signal in the IR780 group decayed rapidly in the systemic circulation due to rapid renal clearance. Figure 6 In vitro organ imaging of the C-D region showed that the fluorescence intensity of tumor tissue in the SLNPs@780 group was 19.19 times that of the IR780 group and 5.48 times that of the Lip@780 group. Moderately elevated liver and kidney signals reflected the uptake and prolonged circulation time characteristics of the nanoparticles via a mononuclear phagocytic system.

[0062] 6. In vivo antitumor effects of SLNPs@780 6.1 Mouse Tumor Simulation Experiment Male BALB / c nude mice (4-6 weeks old, weighing 16-18 g) were subcutaneously inoculated with HCT-116 cells (1×10⁻⁶) one week after acclimatization. 7 (0.2 mL / animal, per tumor). Wait until the tumor volume grows to 50-100 mm. 3 Animals were randomly divided into 6 groups (n=6 / group): (I) PBS control group, (II) oxaliplatin positive control group (OXA, 5 mg / kg), (III) SSD group (2.5 mg / kg), (IV) IR780 group, (V) SLNPs@780 no-light group (IR780 dose 2 mg / kg), and (VI) SLNPs@780+L group (IR780 dose 2 mg / kg). All drugs were administered via tail vein injection, once every 3 days for a total of 3 administrations. Four hours after administration, the tumor sites of mice in group (VI) were irradiated with an 808 nm laser (1 W / cm²). 2 (5 min). Tumor volume and mouse weight were measured every 3 days throughout the treatment period. Tumor volume was calculated using the formula V = 1 / 2 × W. 2 The length of the tumor was calculated as ×L (L is the longest diameter of the tumor, W is the shortest diameter). After treatment, the mice were euthanized, and the tumor tissue was removed and weighed. Results are shown below. Figure 7 Parts A through E in the text.

[0063] 6.2 Tumor histopathological analysis The tumor tissue excised in section 6.1 was fixed with 4% paraformaldehyde, embedded in paraffin, cut into 20 μm sections, and stained with hematoxylin and eosin (H&E). The results are shown in [Figure 1]. Figure 7 The F part in the text.

[0064] Figure 7 The in vivo antitumor effect of SLNPs@780 prepared in Example 2 is shown in Figure A, where A is the actual image of mouse tumors in different treatment groups and the tumor growth curves of all mice, B is an overview of the tumor growth curves in different treatment groups, C is the actual image of ex vivo tumors in different treatment groups, D is the tumor weight in different treatment groups, E is the body weight change curve in different treatment groups, and F is the H&E staining results in different treatment groups. Figure 7The results in sections A through D showed that the tumors in the PBS control group exhibited rapid progressive growth, increasing in volume to approximately 1500 mm. 3 The tumor growth curves of the IR780 group, the SLNPs@780 no-light group, and the Lip@780+L group largely overlapped with those of the PBS control group, indicating that the free photosensitizer lacked targeting. SLNPs@780 showed no anti-tumor activity under no-light conditions, and Lip@780+L produced only a weak therapeutic effect due to the lack of active targeting. Tumor growth was significantly inhibited in the SLNPs@780+L group, with tumor volume reduced by approximately 80% compared to the PBS control group, and the tumor inhibition rate was comparable to that of the oxaliplatin group. Figure 7 The results of Part E showed that during the treatment period, the body weight of mice in the SLNPs@780+L group was not significantly different from that in the PBS group, indicating that the nanoparticles did not cause significant systemic toxicity at the therapeutic dose; mice in the oxaliplatin group had a significant decrease in body weight (approximately 15%) and showed toxic side effects such as reduced activity. Figure 7 The results in the middle F section showed that tumor cells in the PBS control group exhibited typical epithelial-like morphology, with large, deeply stained nuclei and numerous mitotic figures. Tumor tissue in the SLNPs@780+L group showed extensive structural destruction, with significant vacuolation, extensive nuclear pyknosis, and nuclear deletion. These results collectively demonstrate that SLNPs@780-mediated photodynamic therapy effectively achieves targeted tumor delivery.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of saikosaponin D-functionalized lipid nanoparticle, characterized in that, The saikosaponin D functionalized lipid nanoparticles comprise a lipid bilayer and a photosensitizer encapsulated within the lipid bilayer; the lipid bilayer is composed of phospholipids, saikosaponin D, and polyethylene glycol-modified phospholipids.

2. The saikosaponin D-functionalized lipid nanoparticles according to claim 1, characterized in that, The mass ratio of the phospholipid, saikosaponin D, and polyethylene glycol-modified phospholipid is 8:1:

1.

3. The saikosaponin D-functionalized lipid nanoparticles according to claim 1 or 2, characterized in that, The phospholipid is selected from soybean phosphatidylcholine; the polyethylene glycol-modified phospholipid is selected from DSPE-PEG. 2000 .

4. The saikosaponin D-functionalized lipid nanoparticles according to claim 1, characterized in that, The photosensitizer encapsulation rate in the saikosaponin D functionalized lipid nanoparticles is ≥90%, and the drug loading of the photosensitizer is 6.1%.

5. The saikosaponin D-functionalized lipid nanoparticles according to claim 1 or 4, characterized in that, The photosensitizer is selected from photosensitizer IR780.

6. The saikosaponin D-functionalized lipid nanoparticles according to claim 1, characterized in that, The saikosaponin D functionalized lipid nanoparticles have a particle size of 83.54±2.1 nm, a polydispersity index (PDI) of 0.19±0.05, and a zeta potential of -24.77±2.7 mV.

7. A method for preparing saikosaponin D-functionalized lipid nanoparticles according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Dissolve phospholipids, saikosaponin D and polyethylene glycol-modified phospholipids in an organic solvent and mix them evenly to obtain a mixed solution; (2) The photosensitizer is dissolved in dimethyl sulfoxide and then added to the mixed solution obtained in step (1). The organic solvent is then removed by rotary evaporation to obtain a drug-loaded lipid film. (3) Add ultrapure water to the drug-loaded lipid film obtained in step (2) for hydration, then perform probe ultrasonic treatment, and cool to room temperature to obtain the saikosaponin D functionalized lipid nanoparticles.

8. The preparation method according to claim 7, characterized in that, In step (1), the organic solvent is selected from one or more of dichloromethane, methanol, and anhydrous ethanol; In step (2), the temperature of the rotary evaporation is 40~50℃; In step (3), the hydration temperature is 40℃ and the hydration time is 30 min; the power of the probe ultrasonic treatment is 40~60 W and the total time of the probe ultrasonic treatment is 5~10 min.

9. The use of saikosaponin D functionalized lipid nanoparticles according to any one of claims 1 to 6 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is colorectal cancer; the antitumor drug is a photodynamic therapy drug.

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