Pork liver source / photosensitizer-loaded pork liver source lipid nanoparticles and application thereof in endoplasmic reticulum stress enhanced phototherapy

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a pork liver source / photosensitizer-loaded pork liver source lipid nanoparticle and application thereof in endoplasmic reticulum stress enhanced phototherapy. The lipid extraction method provided by the invention has the advantages of mature process, low cost, easily available raw materials, clear components of the obtained lipid, and high biocompatibility. The pork liver source lipid nanoparticles prepared from natural lipid extracted from pork liver have good endoplasmic reticulum targeting property, and can be used as a carrier to efficiently load a photosensitizer IR780; the related nanoparticle preparation method is simple, good in repeatability and easy for expanded production. A phototherapy system constructed by the nanoparticles can generate active oxygen in situ in an endoplasmic reticulum under near-infrared laser irradiation to initiate strong endoplasmic reticulum stress, so that the anti-tumor effect of photothermal and photodynamic combined treatment is remarkably enhanced, and the nanoparticles can be widely applied to efficient and accurate treatment of various solid tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and specifically discloses a pig liver-derived lipid nanoparticle loaded with a photosensitizer and application of the pig liver-derived lipid nanoparticle in endoplasmic reticulum stress-enhanced phototherapy. BACKGROUND

[0002] Traditional cancer treatments such as chemotherapy and radiotherapy have limitations such as strong systemic toxicity, drug resistance and poor targeting. Phototherapy (including photothermal therapy PTT and photodynamic therapy PDT) as a non-invasive alternative solution has attracted widespread attention, but its clinical application still faces two major challenges: (1) insufficient accumulation of phototherapy agents at tumor sites; (2) the antioxidant defense system of tumor cells can weaken the effect of photodynamic therapy.

[0003] To overcome these limitations, existing technologies focus on improving the targeting ability of phototherapy agents and enhancing the intracellular oxidative stress level. The endoplasmic reticulum, as a key organelle for regulating protein folding, calcium homeostasis and lipid synthesis, has become an important target for cancer treatment. Studies have shown that by specifically targeting the endoplasmic reticulum and locally producing reactive oxygen species, strong endoplasmic reticulum stress can be induced, which can effectively induce tumor cell apoptosis when the cell's adaptive capacity is exceeded, thereby significantly enhancing the effect of phototherapy.

[0004] Lipid nanoparticles are ideal drug delivery carriers due to their good biocompatibility, biodegradability and high drug loading capacity. Compared with synthetic lipid nanoparticles, naturally lipid-derived nanoparticles have better biological safety and cell membrane similarity, are more easily internalized by tumor cells and have fewer side effects. However, existing naturally lipid-derived nanoparticles still have some obvious deficiencies, mainly including suboptimal in vivo targeting and biodistribution, insufficient in vivo stability and controllable drug release, weak interference ability with the tumor antioxidant system, and the difficulty in preparing reproducible and standardized nanoparticles due to differences in natural lipid sources, which collectively limit their further application in phototherapy. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies of the prior art and provide a pig liver-derived lipid nanoparticle, a preparation method thereof and application thereof.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a preparation method of a pig liver-derived lipid nanoparticle, comprising the following steps: (1) Lipid extraction: extracting pig liver lipid from pig liver tissue; Specifically comprising: providing pig liver tissue, homogenizing the pig liver tissue with a first organic solvent to obtain a homogenate, and stirring and extracting the homogenate in an ice bath for 1 h; adding a methanol aqueous solution to the mixture after extraction, collecting the supernatant after phase separation, centrifuging the supernatant, collecting the supernatant after centrifugation, and removing the solvent in the supernatant by rotary evaporation to obtain a crude lipid extract; dissolving the crude lipid extract with dichloromethane, and performing nitrogen blowing and vacuum drying treatment to obtain pig liver lipid.

[0007] The first organic solvent is a mixed solvent of methyl tert-butyl ether and methanol, and the volume ratio is 3:1; in the methanol aqueous solution, the volume ratio of methanol to water is 3:1; the mass-volume ratio of the pig liver tissue to the first organic solvent is 1:3-1:5, and the mass-volume ratio of the pig liver tissue to dichloromethane is 1:0.8-1:1.2.

[0008] Preferably, the mass-volume ratio of the pig liver tissue to the first organic solvent is 1:4, the mass-volume ratio of the pig liver tissue to dichloromethane is 1:1, the standing time is 10 min, and the centrifugation condition is 12000 rpm, 4℃, and 10 min.

[0009] The obtained pig liver lipid is composed of the following components in mass percentage: phosphatidylcholine 36%, triglyceride 27%, lysophosphatidylcholine 11%, sphingomyelin 10%, phosphatidylethanolamine 6%, lysophosphatidylethanolamine 2%, ceramide 2%, phosphatidylglycerol 2%, diglyceride 1%, and other lipid components 3%.

[0010] (2) Nanoparticle preparation: the pig liver lipid obtained in step (1) is prepared into a lipid nanoparticle by a reverse solvent method.

[0011] Specifically, the pig liver lipid obtained in step (1) is dissolved with a second solvent; the dissolved mixed solution is quickly injected into pure water under magnetic stirring to form pig liver-derived lipid nanoparticles (LLNPs).

[0012] The nanoparticle of the application can also load a photosensitizer IR780, and the specific method is as follows: the pig liver lipid obtained in step (1) is co-dissolved with the photosensitizer IR780 using a second solvent; the dissolved mixed solution is quickly injected into pure water under magnetic stirring to form pig liver-derived lipid nanoparticles loaded with IR780 (IR780@LLNPs).

[0013] The second solvent is ethanol or a solvent mixed by ethanol and dimethyl sulfoxide at a volume ratio of 4:1; the concentration of the pig liver lipid in the second solvent is 5-20 mg / mL; and the volume ratio of the mixed solution to pure water is 1:9.

[0014] Preferably, the concentration of the pig liver lipid in the second solvent is 10 mg / mL.

[0015] The mass ratio of the pig liver lipid to IR780 is 1:0.005-1:0.04; preferably the mass ratio of the pig liver lipid to IR780 is 1:0.02.

[0016] In a second aspect, the present application provides pig liver-derived lipid nanoparticles obtained by the above preparation method, in particular pig liver-derived lipid nanoparticles loaded with IR780.

[0017] In a third aspect, the present application also provides the use of the above pig liver-derived lipid nanoparticles loaded with IR780 in targeting endoplasmic reticulum of tumor cells.

[0018] The above pig liver-derived lipid nanoparticles loaded with IR780 are used for preparing a drug for treating breast cancer; wherein the drug exerts an anti-tumor effect through a combination of photothermal and photodynamic therapy; and 808 nm near-infrared light is used for irradiation during treatment.

[0019] The present application has the following advantages: (1) High biological safety: the use of pure natural biological source lipid makes the prepared lipid nanoparticles have excellent biocompatibility and biodegradability, and high safety in vivo.

[0020] (2) Synergistic treatment mechanism: the loaded IR780 can produce photothermal effect and photodynamic effect under 808 nm laser irradiation. The photothermal effect directly destroys the endoplasmic reticulum structure, and the photodynamic effect generates a large amount of ROS in situ. The two effects synergistically induce strong and irreversible endoplasmic reticulum stress, leading to tumor cell apoptosis, and achieving a synergistic treatment effect of "1+1>2".

[0021] (3) Unique active targeting ability: the present application finds that the nanoparticles composed of the pig liver natural lipid have natural affinity and targeting ability for the endoplasmic reticulum of tumor cells without any target head modification. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Pies chart of omics analysis of pig liver lipid extracted for Example 1.

[0023] Figure 2 Sample solution graph of LLNPs and IR780@LLNPs prepared for Example 3, Figure 2 A is the sample solution graph of LLNPs, Figure 2 B is the sample solution graph of IR780@LLNPs.

[0024] Figure 3 Particle size determination data graph of LLNPs and IR780@LLNPs for Example 5, Figure 3 A is the particle size determination data graph of LLNPs, Figure 3Figure 6 is a graph of stability of IR780@LLNPs in H2O.

[0025] Figure 4 Figure 7 is a graph of potential measurement data for Example 5 LLNPs and IR780@LLNPs.

[0026] Figure 5 Figure 8 is a graph of stability of IR780@LLNPs in PBS.

[0027] Figure 6 Figure 9 is a graph of stability of IR780@LLNPs in FBS.

[0028] Figure 7 Figure 10 is a graph of stability of IR780@LLNPs in serum.

[0029] Figure 8 Figure 11 is a graph of temperature ramping curve of IR780@LLNPs.

[0030] Figure 9 Figure 12 is a graph of IR780@LLNPs in vivo distribution experiment results in 4T1 cells.

[0031] Figure 10 Figure 13 is a graph of IR780@LLNPs cytotoxicity evaluation in 4T1 cells.

[0032] Figure 11 Figure 14 is a graph of IR780@LLNPs reactive oxygen species experiment results under laser irradiation.

[0033] Figure 12 Figure 15 is a graph of IR780@LLNPs real-time fluorescence imaging in mice.

[0034] Figure 13 Figure 16 is a graph of tumor volume change in mice during IR780@LLNPs treatment.

[0035] Figure 14 Figure 17 is a graph of body weight change in mice during IR780@LLNPs treatment.

[0036] Figure 15 Figure 18 is a graph of H&E staining of tumor sections of mice in different treatment groups of IR780@LLNPs.

[0037] Figure 16 Figure 19 is a graph of hemolysis data of IR780@LLNPs. DETAILED DESCRIPTION

[0038] The present application will be further described below with reference to the accompanying drawings and specific examples. Those skilled in the art will be able to implement the present application based on these descriptions, and in addition, the examples of the present application described in the following description are generally only a part of the examples of the present application, and not all the examples. Therefore, all other examples obtained by those skilled in the art based on the examples in the present application without creative work shall fall within the scope of protection of the present application.

[0039] In the following examples, the experimental methods described are the commonly used methods in the art, and the experimental materials and reagents described are the commonly used materials and reagents in the art, unless otherwise specified.

[0040] Example 1: Extraction of Lipids

[0041] (1) 5 g of pig liver tissue was homogenized with 20 mL of a mixture of methyl tert-butyl ether and methanol (MTBE:MeOH=3:1, V / V), and the mixture was extracted by stirring in an ice bath for 1 h.

[0042] (2) 13 mL of a mixture of methanol and water (MeOH:H2O=1:3, V / V) was added to the product of step (1), and the phases were allowed to separate by standing for 10 min. The supernatant was collected by centrifugation at 12000 rpm and 4°C for 10 min, and the crude product was obtained by rotary evaporation.

[0043] (3) The crude product of step (2) was redispersed with 5 mL of dichloromethane, and after nitrogen purging to remove the dichloromethane, vacuum drying was performed for 24 h. The extracted pig liver lipids were stored at -80°C for later use.

[0044] The specific components of the extracted pig liver lipids were detected by liquid chromatography-mass spectrometry. The specific steps are as follows: 10 mg of lipids were redissolved with 2 mL of dichloromethane, 800 μL of the sample was evaporated to dryness, 300 μL of isopropyl alcohol-methanol (V:V=1:1, containing a mixed internal standard, 4 μg / mL) was added for redissolution, vortexed for 30 s, ultrasonic extraction for 10 min, and -40°C overnight standing; centrifugation for 10 min (12000 rpm, 4°C), 150 μL of supernatant was taken and loaded into an LC-MS sample vial for LC-MS analysis. Figure 1The image shows the compositional analysis of lipids in pig liver. The results indicate that pig liver contains phosphatidylcholine (PC) (~36% of total lipids), triglycerides (TG) (~27% of total lipids), lysophosphatidylcholine (LPC) (~11% of total lipids), sphingomyelin (SM) (~10% of total lipids), phosphatidylethanolamine (PE) (~6% of total lipids), lysophosphatidylethanolamine (LPE) (~2% of total lipids), ceramide (Cer) (~2% of total lipids), phosphatidylglycerol (PG) (~2% of total lipids), diglycerides (DG) (~1% of total lipids), and other lipid components (~3% of total lipids).

[0045] Example 2: Optimization Experiment

[0046] To verify the method for extracting lipids from pig liver, various parameter optimization experiments were conducted. Except for changing the material-liquid ratio (W / V) of pig liver and MTBE:MeOH and the material-liquid ratio (W / V) of pig liver and dichloromethane, the experimental steps and conditions were the same as in Example 1. The specific results are shown in Tables 1-2.

[0047] Table 1. Effect of the solid-liquid ratio of porcine liver and MTBE:MeOH (W / V) on lipid extraction yield.

[0048] When the solid-liquid ratio was 1:3, although the solvent could completely cover the pig liver tissue, the extraction yield was only 0.0668g, indicating insufficient solvent. Increasing the ratio to 1:4 significantly increased the extraction yield to 0.1189g, demonstrating a significant improvement in extraction efficiency. Further increasing the solvent ratio to 1:5 only slightly increased the extraction yield to 0.1214g, indicating that extraction at the 1:4 ratio was approaching saturation. Therefore, selecting a solid-liquid ratio of 1:4 for subsequent experiments ensures a high extraction rate while effectively controlling solvent usage and reducing the load on subsequent processing.

[0049] Table 2. Effect of the weight-to-liquid ratio (W / V) of porcine liver and dichloromethane on lipid extraction yield.

[0050] Increasing the amount of dichloromethane helps to fully extract lipids from pig liver. The amount of dichloromethane used is positively correlated with lipid extraction, reaching saturation at a 1:1 ratio. To control solvent usage, a 1:1 solid-liquid ratio was ultimately chosen for subsequent experiments.

[0051] Example 3: Preparation of LLNPs and IR780@LLNPs

[0052] Take the lipid prepared in Example 1, add anhydrous ethanol to dissolve and configure into a lipid ethanol solution with a concentration of 10 mg / mL; under the condition of continuous stirring at 1500 rpm of a magnetic stirrer, inject 1 mL of the above lipid ethanol solution into 9 mL of deionized water quickly, continue stirring for 2 min, and prepare pig liver-derived lipid nanoparticles (LLNPs).

[0053] Take 10 mg of the lipid prepared in Example 1 and 0.2 mg of IR780, dissolve them in anhydrous ethanol to obtain an IR780-lipid mixed ethanol solution with a concentration of 10 mg / mL; according to the above preparation conditions of LLNPs (i.e., under the condition of continuous stirring at 1500 rpm of a magnetic stirrer, inject 1 mL of the mixed ethanol solution into 9 mL of deionized water quickly, continue stirring for 2 min), prepare IR780-loaded pig liver-derived lipid nanoparticles (IR780@LLNPs). As Figure 2 The sample images show that the prepared LLNPs and IR780@LLNPs have good dispersibility, no precipitation and separation phenomenon.

[0054] Example 4: Preferred experiment

[0055] To explore the effect of lipid concentration on nanoparticles, the lipid obtained in Example 1 was dissolved in anhydrous ethanol to different concentrations, 1 mL of the lipid ethanol solution was injected into 9 mL of deionized water, and the mixture was stirred at 1500 rpm for 2 min, finally obtaining LLNPs with different concentrations. The particle size and dispersion coefficient of each group were detected, and the effect of pig liver lipid concentration on the particle size of lipid nanoparticles is shown in Table 3.

[0056] Table 3 Effect of pig liver lipid concentration on the particle size of lipid nanoparticles

[0057] When the lipid concentration is 10 mg / mL, a key balance between system stability and drug loading potential can be achieved. Compared with lower concentrations, this concentration can provide more carrier materials to improve drug loading efficiency, and compared with higher concentrations, this concentration can effectively avoid the stability risks caused by particle aggregation and uneven distribution. Therefore, the concentration of 10 mg / mL is the most stable and reliable choice for subsequent research.

[0058] To explore the effect of IR780 concentration on the nanoparticles, the lipids prepared in Example 1 were mixed with different amounts of IR780, and dissolved in anhydrous ethanol to obtain an IR780-lipid mixed ethanol solution with a concentration of 10 mg / mL. 1 mL of the IR780-lipid mixed ethanol solution was quickly injected into 9 mL of deionized water, and the mixture was stirred at 1500 rpm for 2 min. Finally, IR780@LLNPs with different IR780 concentrations were obtained. The particle size and dispersion coefficient of each group were detected. The effect of IR780 concentration on the particle size of the lipid nanoparticles is shown in Table 4.

[0059] Table 4 Effect of IR780 concentration on the particle size of the lipid nanoparticles

[0060] With the increase of IR780 concentration, the particle size and PDI of the nanoparticles changed. Considering the drug loading and the quality of the nanoparticles, a mass ratio of 1:0.02 was selected for subsequent experiments, which could provide sufficient IR780 loading and ensure the system had a suitable particle size and excellent monodispersity, laying a reliable foundation for subsequent biological applications.

[0061] Example 5: Measurement of particle size and potential of LLNPs and IR780@LLNPs

[0062] The LLNPs and IR780@LLNPs prepared in Example 3 were used to measure the size and potential of the samples by dynamic light scattering method.

[0063] As shown in Figure 3 , the particle size of LLNPs was 71.99 nm, and the PDI was 0.229. The particle size of IR780@LLNPs was 69.95 nm, and the PDI was 0.216, with good dispersity. As shown in Figure 4 , the Zeta potential measurement result of LLNPs was -40.17 ± 0.68 mV, and the Zeta potential measurement result of IR780@LLNPs was -35.77 ± 1.10 mV.

[0064] Example 6: Stability of IR780@LLNPs

[0065] To evaluate the stability of IR780@LLNPs in a physiological environment, the dispersion of the IR780@LLNPs in different media was investigated. The IR780@LLNPs prepared in Example 3 were placed in H2O, PBS and FBS, respectively, and diluted to a lipid concentration of 100 μg / mL. The particle size and polydispersity index (PDI) changes within five days were tested by Malvern particle potential instrument to evaluate the stability. The results are shown in Figures 5-7As shown, the particle size of IR780@LLNPs changed little in different environments, and the PDI remained below 0.3, indicating that it had good dispersibility and stability. This stability ensured the reliability of IR780@LLNPs in in vivo applications, reducing the risk of aggregation and deposition, and helping to improve its bioactivity and safety in vivo.

[0066] Example 7: In vitro photothermal effect of IR780@LLNPs

[0067] To explore the in vitro photothermal effect of IR780@LLNPs, IR780@LLNPs with IR780 concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL were irradiated with an 808 nm laser for 120 s, with a power density of 1.5 W / cm 2 The real-time temperature of the sample was recorded by an infrared thermal imager, and the temperature rise curve was plotted. As shown in Figure 8 After irradiation, the temperatures of IR780@LLNPs with IR780 concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL reached 32.6℃, 37.8℃, 49.6℃, and 64.4℃, respectively, with temperature increases of 4.7℃, 10.8℃, 22.1℃, and 36.8℃, respectively, indicating that IR780@LLNPs had good photothermal properties and potential for photothermal therapy as an anti-tumor drug.

[0068] Example 8: Intracellular distribution of IR780@LLNPs

[0069] The distribution of coumarin 6-labeled IR780@LLNPs in mouse breast cancer cells (4T1) was observed using a confocal laser microscope. 4T1 cells were evenly distributed in a confocal culture dish at a seeding density of 2×10 5 cells per well, and then transferred to a incubator at 37℃ and 5% CO2 for constant temperature incubation for 24 h. Diluted coumarin 6-labeled IR780@LLNPs (IR780 concentration of 0.5 μg / mL) were added to each well and incubated for 4 h. After incubation, the cells were stained with ER-TrackerTM Red (1 μM) for 30 min, fixed with a 4% paraformaldehyde solution for 10 min, and then the cell nuclei were stained with DAPI (10 μg / mL) for 10 min. The cells were then imaged using a confocal laser microscope. As shown in Figure 9 IR80@LLNPs were widely accumulated in the endoplasmic reticulum of 4T1 cells.

[0070] Example 9: In vitro therapeutic effect of IR780@LLNPs

[0071] The cytotoxicity of IR780@LLNPs on 4T1 cells was evaluated by MTT method. 4T1 cells were seeded in 96-well plates at a density of 1 × 10 4 For the non-laser irradiation group, the cells were incubated with Free IR780 or IR780@LLNPs for 24 h. For the near-infrared laser irradiation group, the cells were incubated with Free IR780 or IR780@LLNPs for 4 h, and then irradiated with an 808 nm laser with a power of 1.5 W / cm 2 After 3 min of irradiation, the cells were incubated for a total of 24 h, and then the supernatant was discarded. After adding MTT reagent and incubating for another 4 h, the absorbance was measured at a wavelength of 490 nm using a microplate reader. As shown in Figure 10 , the tumor cell viability of the Free IR780 + Laser and IR780@LLNPs + Laser groups showed a certain concentration-dependent decrease, and in particular, the IR780@LLNPs + Laser group showed the highest tumor killing ability.

[0072] Example 10: Experiment on the generation of reactive oxygen species by IR780@LLNPs under laser irradiation

[0073] 4T1 cells were evenly distributed in confocal culture dishes at a seeding density of 2 × 10 5 cells per well, and then transferred to an incubator at 37°C and 5% CO2 for constant incubation for 24 h. Diluted IR780@LLNPs (IR780 concentration of 0.5 μg / mL) were added to each well and incubated for 4 h. After dark treatment or laser treatment, 4T1 cells were labeled with 10 μM DCFH-DA at 37°C for 30 min, and then observed under a confocal microscope. As shown in Figure 11 , compared with Free IR780, IR780@LLNPs generated higher levels of ROS under laser irradiation, indicating that IR780@LLNPs have the potential to locally generate ROS, induce endoplasmic reticulum stress, and enhance the therapeutic effect through the endoplasmic reticulum-related apoptosis pathway.

[0074] Example 11: In vivo biodistribution evaluation of IR780@LLNPs

[0075] The mice used in this example were female Balb / c mice, 16-20 g, 4-6 weeks old. A suspension containing about 2 × 10 6 4T1 cells was subcutaneously injected into the dorsal region of Balb / c mice to establish a 4T1 tumor-bearing mouse model. When the tumor grew to about 100 mm 3 , subsequent experiments were performed.

[0076] To investigate the distribution of IR780@LLNPs in vivo in mice, 4T1 tumor-bearing mice were injected with saline, Free IR780 and IR780@LLNPs (the dose of IR780 injected into each mouse was 2 mg / kg) via the tail vein, respectively. At 4, 8, 24, 48 and 72 h after injection, the tissue distribution in mice was observed by small animal live imaging. As shown in FIG. 8, compared with Free IR780, IR780@LLNPs had stronger fluorescence signals at the tumor site, indicating stronger tumor targeting ability, accumulation ability and longer retention time. This indicates that IR780@LLNPs achieve more effective accumulation at the tumor site. Figure 12

[0077] Example 12: In vivo therapeutic effect of IR780@LLNPs

[0078] The 4T1 tumor-bearing mice were randomly divided into four groups (5 mice in each group): Saline group, IR780@LLNPs group, Free IR780+Laser group and IR780@LLNPs+Laser group. Three treatments were performed on the 1st, 4th and 7th days, and laser irradiation was performed on the 2nd, 5th and 8th days in the laser group. The tumor volume and body weight were measured every two days for 16 days. As shown in FIG. 9, the results showed that the tumor volume of all non-laser irradiation groups increased rapidly within 16 days; however, the tumor growth of the near-infrared laser irradiation groups was inhibited. Among them, the tumor relative volume of the IR780@LLNPs+Laser group was the smallest, and the tumor inhibition effect was the strongest. Figure 13 Figure 14 As shown in FIG. 10, no detectable changes in the body weight of mice were observed during the treatment period. As shown in FIG. 11, for the H&E staining results, the cell nucleus rupture and disappearance of the IR780@LLNPs+Laser group were the most serious compared with the Saline group, indicating the strongest killing effect on tumor cells. Figure 15

[0079] Example 13: In vivo toxicological analysis of IR780@LLNPs

[0080] ​​​The hemocompatibility of IR780@LLNPs was evaluated using hemolysis test. Briefly, fresh whole blood was collected from the orbital plexus of mice and the supernatant was removed. The precipitated red blood cells were washed with PBS. Subsequently, the red blood cells were added to different concentrations of IR780@LLNPs. Deionized water and PBS were used as positive and negative controls, respectively. The mixture was incubated at 37 °C for 2 h. After incubation, the mixture was centrifuged at 1000 rpm for 10 min. The mixture was then photographed, and the absorbance of the supernatant at 540 nm was measured using a microplate reader. The hemolysis ratio was calculated using the following formula, hemolysis ratio (%) = (As-An) / (Ap-An) x 100%, where As, Ap, and An represent the absorbance of the sample, positive control, and negative control, respectively. As shown in Figure 16 FIG. 21, the hemolysis ratio of IR780@LLNPs was less than 5%, indicating that the IR780@LLNPs prepared in Example 2 had good biocompatibility and safety at a lipid concentration below 200 pg / mL. In summary, IR780@LLNPs have very low risk of red blood cell hemolysis and potential clinical application prospects in anticancer.

Claims

1. A method for preparing porcine liver-derived / photosensitizer-loaded porcine liver-derived lipid nanoparticles, characterized in that, The preparation method steps are as follows: (1) Lipid extraction: Extraction of pig liver lipids from pig liver tissue; (2) Nanoparticle preparation: The pig liver lipid obtained in step (1) or the pig liver lipid obtained in step (1) and photosensitizer are prepared into lipid nanoparticles by antisolvent method.

2. The method for preparing lipid nanoparticles according to claim 1, characterized in that, In step (1), the lipid extraction method is as follows: pig liver is homogenized with a first organic solvent to obtain a homogenate, and the homogenate is extracted by stirring in an ice bath for 1 hour; methanol aqueous solution is added to the extracted mixture, and after standing and phase separation, the supernatant is collected, the supernatant is centrifuged, the supernatant after centrifugation is collected and the solvent is removed by rotary evaporation to obtain crude lipid extract; the crude lipid extract is dissolved in dichloromethane, and after nitrogen purging and vacuum drying, pig liver lipid is obtained.

3. The method for preparing lipid nanoparticles according to claim 2, characterized in that, The first organic solvent is a mixture of methyl tert-butyl ether and methanol in a volume ratio of 3:1; the mass-volume ratio of pig liver tissue to the first organic solvent is 1:3-1:5; in the methanol-water solution, the volume ratio of methanol to water is 3:1; and the mass-volume ratio of pig liver tissue to dichloromethane is 1:0.8-1:1.

2.

4. The method for preparing lipid nanoparticles according to claim 2, characterized in that, The obtained porcine liver lipids consist of the following components by weight percentage: phosphatidylcholine 36%, triglycerides 27%, lysophosphatidylcholine 11%, sphingomyelin 10%, phosphatidylethanolamine 6%, lysophosphatidylethanolamine 2%, ceramide 2%, phosphatidylglycerol 2%, diglycerides 1%, and other lipid components 3%.

5. The method for preparing lipid nanoparticles according to claim 2, characterized in that, Step (2) The preparation method of nanoparticles is as follows: dissolve porcine liver lipids in a second solvent, or dissolve porcine liver lipids and photosensitizer IR780 together in a second solvent; under magnetic stirring, quickly inject the dissolved mixed solution into pure water to form porcine liver-derived lipid nanoparticles LLNPs or porcine liver-derived lipid nanoparticles IR780@LLNPs loaded with IR780.

6. The method for preparing lipid nanoparticles according to claim 5, characterized in that, The second solvent is ethanol or a mixture of ethanol and dimethyl sulfoxide at a volume ratio of 4:1; the volume ratio of the mixed solution to pure water is 1:9; the concentration of porcine liver lipids in the second solvent is 5-20 mg / mL; the mass ratio of porcine liver lipids to IR780 is 1:0.005-1:0.

04.

7. A porcine liver-derived / photosensitive agent-loaded porcine liver-derived lipid nanoparticle prepared according to the method of claim 1.

8. The application of porcine liver-derived lipid nanoparticles loaded with photosensitizer prepared according to the method of claim 1, characterized in that, The nanoparticles are used to prepare agents that target the endoplasmic reticulum of tumor cells.

9. The application of porcine liver-derived lipid nanoparticles loaded with photosensitizer according to claim 8, characterized in that, The tumor cells are breast cancer cells.

Images