A lipid assembly loaded with 5-aminolevulinic acid, its preparation method and application

Abstract

This invention discloses a lipid assembly loaded with 5-aminolevulinic acid and its preparation method. The lipid assembly is a hollow sphere with a core consisting of a hydrophilic phospholipid-chelating agent conjugate formed by chelation with a reactive phospholipid PEG reagent. The outer layer of the phospholipid-chelating agent conjugate is loaded with 5-aminolevulinic acid via hydrazone bonds, and then combined with a radionuclide as an internal light source excitation photosensitizer. The lipid assembly is loaded with 5-ALA via hydrazone bonds. Utilizing the acid-sensitive bond-breaking characteristics of hydrazone bonds and the prodrug properties of 5-ALA, combined with a radionuclide labeled on the surface of the lipid assembly as an internal light source excitation photosensitizer, this invention can overcome the limitations of external light penetration depth in traditional photodynamic therapy, achieving deep PDT treatment. Combined with chemotherapy drugs, it can minimize the toxic side effects on normal tissues during the combined use of radionuclides and photosensitizers in targeted therapy.

Description

Technical Field

[0001] This invention relates to a lipid assembly, its preparation and application, and more particularly to a lipid assembly loaded with 5-aminolevulinic acid, its preparation and application. Background Technology

[0002] Photodynamic therapy (PDT), compared to traditional chemotherapy and surgery, boasts high spatiotemporal selectivity, low invasiveness, and no drug resistance, making it a novel approach to cancer treatment. However, most clinically approved photosensitizers can only be excited by ultraviolet or visible light. Due to tissue absorption and scattering of light, the penetration depth of common ultraviolet or visible light into tissues is limited to the μm level. This limited penetration depth significantly restricts the clinical application of PDT, and the rapid attenuation of light within tissues often confines PDT treatment to the body surface.

[0003] Cherenkov radiation is a luminescent phenomenon produced when charged particles travel at speeds exceeding the speed of light in a medium. Essentially, it is the polarization of atoms or molecules in the medium caused by charged particles passing through it. The energy of Cherenkov radiation is primarily concentrated in the ultraviolet region, matching the Soret band of most clinically used photosensitizers such as ALA, temoporophyne, and Ce6. Therefore, using Cherenkov radiation as an internal light source to induce photodynamic therapy can overcome the limitations of traditional photodynamic therapy (PDT) in terms of penetration depth. However, during prolonged in vivo circulation, Cherenkov radiation-induced photodynamic therapy (CR-PDT) inevitably causes unnecessary toxic side effects on normal tissues. Furthermore, the poor biodistribution and pharmacokinetic properties of some photosensitizers can severely affect the targeting and therapeutic efficiency of CR-PDT. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a lipid assembly loaded with 5-aminolevulinic acid that is targeted for release in a tumor microacidic environment based on the Cherenkov effect. The second purpose is to provide a method for preparing and applying the above-mentioned lipid assembly.

[0005] Technical solution: The lipid assembly of the present invention includes a phospholipid-chelating agent conjugate formed by chelation reaction of reactive phospholipid PEG reagent and metal chelating agent. The phospholipid-chelating agent conjugate is a hollow sphere, the outer shell of the sphere is a hydrophobic bilayer, and the inner shell of the sphere is a hydrophilic bilayer. The lipid assembly also includes 5-aminolevulinic acid and a radionuclide loaded in the hydrophobic bilayer by hydrazone bonds.

[0006] Preferably, the phospholipid-chelating agent conjugate is as shown in Formula 1, and the reactive phospholipid PEG reagent is DSPE-PEG2000-NH2, wherein Ch is a metal chelating agent.

[0007]

[0008] Preferably, the chelating agent is one of deferoxamine, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-N,N',N”-triacetic acid, 1,4,8,11-tetra(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane, 1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetraazacyclotetradecane, 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15) or 11,13-triene-3,6,9-triacetic acid.

[0009] Preferably, the hydrophobic bilayer is loaded with a hydrophobic chemotherapeutic drug, and the hydrophilic bilayer is loaded with a water-soluble chemotherapeutic drug.

[0010] The preparation method of the above lipid assemblies includes the following steps:

[0011] (1) Dissolve the reactive phospholipid PEG reagent in dimethyl sulfoxide, then add a metal chelating agent, and after the reaction is completed, dialyze and freeze dry to obtain the phospholipid-chelating agent conjugate.

[0012] (2) The phospholipid-chelating agent complex was dissolved in anhydrous dichloromethane solvent, and triethylamine and triphosgene were added dropwise to react and evaporate by rotary evaporation to obtain the intermediate Lipid-COCl; then Lipid-COCl was added dropwise to a dichloromethane solution of NHNH2·H2O, and the reaction was carried out by washing and centrifugation to obtain the Lipid-NHNH2 complex; finally, an ethanol solution of aminolevulinic acid hydrochloride was added dropwise, glacial acetic acid was added dropwise to react in the dark, n-hexane was added, and hydrazone coupling was carried out to obtain the Lipid-ALA complex;

[0013] (3) Dissolve dipalmitoylphosphatidylcholine, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate and Lipid-ALA conjugate in an organic solvent, and extrude the lipid assembly ALA-Lipo by thin film hydration method.

[0014] (6) The lipid assembly ALA-Lipo was mixed with a radionuclide solution for labeling to obtain the radioactive lipid assembly R-ALA-Lipo, which was a radioactive lipid assembly loaded with 5-aminolevulinic acid.

[0015] Preferably, the lipid assembly ALA-Lipo obtained in step (3) is loaded with a hydrophobic chemotherapeutic drug or a water-soluble chemotherapeutic drug; further, specifically: dipalmitoylphosphatidylcholine, cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate, Lipid-ALA conjugate and hydrophobic chemotherapeutic drug are dissolved in an organic solvent, and the lipid assembly ALA-Lipo loaded with hydrophobic chemotherapeutic drug is obtained by film hydration method and extrusion; or dipalmitoylphosphatidylcholine, cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate and Lipid-ALA conjugate are dissolved in an organic solvent, and after film formation, they are hydrated with a buffer containing water-soluble chemotherapeutic drug and extruded to obtain a lipid assembly ALA-Lipo loaded with water-soluble chemotherapeutic drug.

[0016] Preferably, in step (1), the mass ratio of the reactive phospholipid PEG reagent to the metal chelating agent is 5:1 to 3.

[0017] Preferably, in step (2), the mass ratio of the phospholipid Lyso PC, triethylamine and triphosgene is 50:0.012-0.019:150-300, the mass ratio of Lipid-COCl to NHNH2·H2O is 50:44-65, and the mass ratio of Lipid-NHNH2 conjugate to aminolevulinic acid hydrochloride is 50:15-30.

[0018] Preferably, the specific synthetic route for step (2) is as follows:

[0019]

[0020] Preferably, in step (4), the molar ratio of dipalmitoylphosphatidylcholine, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate, and phospholipid-ALA conjugate is 41-42:26-27:1.2:2.0:28-29, and the mass ratio of hydrophobic or water-soluble chemotherapeutic drugs to liposome assemblies is 5-10:100.

[0021] Preferably, in step (6), the radioactive metal nuclide is 89Zr, 177Lu, 68Ga or 64Cu, and the 1 mL lipid assembly ALA-Lipo can react with a radioactive nuclide solution of 0.1 to 1 mCi and be stably labeled.

[0022] The application of the above-mentioned lipid assemblies in the preparation of targeted drugs is to load hydrophobic chemotherapy drugs into the hydrophobic bilayer on the outer side of the lipid assembly shell and / or the hydrophilic bilayer on the inner side of the shell with water-soluble chemotherapy drugs.

[0023] Invention Principle: This invention synthesizes a phospholipid-ALA conjugate via an organic coupling reaction. 5-Aminolevulinic acid (5-ALA) is linked to the phospholipid via a hydrazone bond. Utilizing the acid-sensitive nature of the hydrazone bond, 5-ALA can be released in the slightly acidic environment of tumors. This lipid assembly exhibits approximately 40% 5-ALA release at pH 6.5, approximately 80% release at pH 5.5, and less than 20% release at the normal physiological pH of 7.4. Finally, the lipid assembly was synthesized using a thin-film hydration method with a molar ratio of 41.4:26.6:1.2:2.0:28.8 for DPPC, cholesterol, DSPE-PEG2000, Lipid-DFO, and Lipid-ALA. The synthesized lipid assembly... 89 Zr at a dose of 10 μCi and a 5-ALA concentration greater than 86 μmol / L effectively killed MC38 cells. In treating MC38 tumor-bearing mice, compared to the control group, this lipid assembly effectively inhibited tumor growth over a two-week treatment period. Combined with an internal light source to excite a photosensitizer containing a radionuclide, a radionuclide-labeled phospholipid-chelating agent conjugate was obtained. This conjugate, assembled with other phospholipid components, can be used for labeling various metallic radionuclides, such as… 89 Zr、 177 Lu、 68 Ga or 64 Cu, with a labeling rate greater than 99%, exhibits excellent labeling stability in serum-containing media. Furthermore, this lipid assembly can be used to co-load hydrophilic or hydrophobic chemotherapeutic drugs, thereby achieving combination therapy and further enhancing the efficacy of tumor treatment.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The radionuclide labeled on the surface of the lipid assembly is used as an internal light source to excite photosensitizer, which can break through the limitation of the tissue penetration depth of the external light in the traditional photodynamic therapy process and realize deep PDT therapy; (2) The lipid assembly is loaded with 5-ALA through hydrazone bond coupling. It utilizes the acid-sensitive bond breaking characteristics of hydrazone bond and the prodrug characteristics of 5-ALA to release it in a specific tumor micro-acid environment. The release amount is 2 to 4 times higher than that of the prior art. Therefore, the lipid assembly will only produce PDT therapy effect at the tumor site, thereby avoiding the toxic side effects on normal tissues in the process of using radionuclide and photosensitizer in combination to the greatest extent. Attached Figure Description

[0025] Figure 1 For the intermediate Lipid-COCl 1 H NMR spectrum;

[0026] Figure 2 For Lipid-NHNH21 H NMR spectrum;

[0027] Figure 3 For product Lipid-ALA 1 H NMR spectrum;

[0028] Figure 4 Infrared spectra of Lipid-COCl, Lipid-NHNH2, and Lipid-ALA;

[0029] Figure 5 TEM image of the lipid assembly ALA-Lipo;

[0030] Figure 6 A schematic diagram showing the particle size change of the lipid assembly ALA-Lipo under different conditions over 48 hours;

[0031] Figure 7 Schematic diagram of ALA-Lipo drug release from lipid assemblies under different conditions;

[0032] Figure 8 Lipid assemblies under different conditions 89 Schematic diagram of the stability of Zr-ALA-Lipo radiochemical labeling;

[0033] Figure 9 A schematic diagram showing the singlet oxygen production of different components;

[0034] Figure 10 For different cells to 89 A diagram illustrating Zr-ALA-Lipo uptake;

[0035] Figure 11 for 89 A schematic diagram illustrating the in vitro therapeutic effects of Zr-ALA-Lipo;

[0036] Figure 12 A schematic diagram showing the ROS generation of different components;

[0037] Figure 13 AM / PI double staining images of live and dead cells from different components;

[0038] Figure 14 A schematic diagram of tumor growth curves for different components;

[0039] Figure 15 A schematic diagram showing the changes in mouse body weight for different components;

[0040] Figure 16 This is a schematic diagram of the morphological structure of a lipid assembly loaded with 5-aminolevulinic acid. Detailed Implementation

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

[0042] Example 1

[0043] Synthesis of the phospholipid-chelating agent conjugate: 10 mg of DSPE-PEG2000-NH2 was dissolved in dimethyl sulfoxide (DMSO), and 3 eq of deferoxamine (DFO) was added to the solution. The mixture was stirred overnight at room temperature. Unreacted chelating agent was then removed by dialyzing (1000 Da, MW), and finally, the phospholipid-chelating agent conjugate was obtained by lyophilization.

[0044] Example 2

[0045] Synthesis of Lipid-ALA:

[0046] (1) Synthesis of Lipid-COCl: Under anhydrous conditions, 50 mg of phospholipid Lyso PC was dissolved in 5 ml of anhydrous DCM and placed in a 100 ml round-bottom flask. Then, 1.2 eq of triethylamine (TEA) was added dropwise to the reaction mixture under ice bath conditions. Simultaneously, 10 eq of triphosgene was diluted in anhydrous DCM and then added dropwise to the reaction mixture, and the mixture was stirred at room temperature. Since HCl gas is generated during the reaction, a gas absorption device with NaOH solution is required. The reaction was stopped when no more gas was generated. The product was rotary evaporated with anhydrous DCM more than three times to remove excess triphosgene. Finally, Lipid-COCl was obtained.

[0047] (2) Synthesis of phospholipid-hydrazine conjugate (Lipid-NHNH2): 100 mg of Lipid-COCl was dissolved in an ice bath and slowly added dropwise to a DCM solution of NHNH2·H2O. After the addition was complete, the reaction mixture was stirred at room temperature for 10 hours. A gas absorption device with NaOH solution was also required to absorb the HCl gas generated during the reaction. After the reaction was completed, the product was washed with n-hexane, centrifuged (5000 r / min) for 5 minutes, and the supernatant was removed. This process was repeated three times to obtain Lipid-NHNH2.

[0048] (3) Synthesis of phospholipid-ALA conjugate (Lipid-ALA): 30 mg of ALA·HCl was dissolved in anhydrous ethanol and then added dropwise to a Lipid-NHNH2 solution. A few drops of glacial acetic acid were added to the reaction solution, and the reaction was stirred overnight at room temperature in the dark. Then, n-hexane was added to the reaction solution, precipitating a large amount of pale yellow solid. After centrifugation and removal of the supernatant, a yellow viscous product (Lipid-ALA) was obtained. Lipid-ALA was placed in a vacuum drying oven overnight to remove residual organic solvents.

[0049] The synthesis roadmap is shown below:

[0050]

[0051] Characterization of Lipid-ALA:

[0052] (1) Proton NMR spectrum

[0053] Weigh 15 mg of the sample to be tested and dissolve it in... 1 Add d DMSO to the NMR tube for testing. 1 H NMR (300 Hz) was used to determine whether the product structure was correct.

[0054] Figure 1 , Figure 2 as well as Figure 3 These are the intermediates Lipid-COCl, Lipid-NHNH2, and the product Lipid-ALA, respectively. 1 The HNMR spectra were obtained, and the corresponding absorption peaks were assigned. The number of hydrogen nuclei and the chemical shift number of the two intermediates and the product Lipid-ALA were consistent with the structure, confirming that they conform to the structure of the target compound.

[0055] (2) Infrared absorption spectrum

[0056] Figure 4 These are the infrared absorption spectra of the intermediates Lipid-COCl and Lipid-NHNH2, and the product Lipid-ALA. In Lipid-COCl, because the free hydroxyl groups of Lipid are substituted, no hydroxyl stretching vibration is observed (broad peak, 3500–4000 cm⁻¹). -1 Lipid-COCl, Lipid-NHNH2, and the product Lipid-ALA all have a range of 1900-1600 cm⁻¹. -1 A strong C=O stretching absorption band appears in the region. Due to inductive and conjugation effects, the C=O stretching absorption bands of Lipid-COCl and Lipid-ALA shift towards longer wavenumbers, while those of Lipid-NHNH2 shift towards shorter wavenumbers. Lipid-NHNH2 exhibits a strong absorption band in the 3300-3500 cm⁻¹ region. -1 A characteristic double peak of primary amines appears between the two peaks, and Lipid-ALA shows a peak at 1690-1640 cm⁻¹. -1 The appearance of C=N stretching absorption bands and in the 3000-3600 cm⁻¹ range -1 A stretching absorption band of carboxylic acid OH was observed. These results confirm the structures of intermediates Lipid-COCl and Lipid-NHNH2, as well as the product Lipid-ALA, and are consistent with... 1 The H NMR spectra corroborate each other.

[0057] Example 3:

[0058] Preparation method of lipid assembly ALA-Lipo:

[0059] The phospholipids were dissolved in a chloroform:methanol mixture at a molar ratio of 41.4:26.6:1.2:2.0:28.8 (DPPC:cholesterol:DSPE-PEG2000:phospholipid-chelating agent conjugate:Lipid-ALA). The phospholipid mixture was placed in a 25 mL round-bottom flask and rotary evaporated at 40 °C in a water bath to form a membrane. The resulting phospholipid membrane was then vacuum-dried overnight to remove residual organic solvent. 3-5 mL of HEPES buffer (0.5 M, pH 7.0) was added, and the membrane was hydrated at 50 °C for 1 h. The membrane was then extruded more than 5 times using a 0.1 μm polycarbonate extruder, filtered through a 0.22 μm filter, and ultrafiltered and centrifuged (3000 rpm, 30 min) to obtain the purified lipid assembly ALA-Lipo. The TEM morphology of the obtained lipid assembly ALA-Lipo is shown below. Figure 5 As shown, it has a spherical structure of uniform size.

[0060] Example 4:

[0061] Stability assay of the lipid assembly ALA-Lipo:

[0062] During incubation for 48 hours in PBS buffer and culture medium (DMEM containing 10% serum) at pH 7.4, pH 6.5, and pH 5.5, the hydrated particle size of ALA-Lipo lipid assemblies was measured at the corresponding time points (Litesizer). TM 500, Anton Paar, Austria).

[0063] The particle size of ALA-Lipo changes with incubation time under different environmental conditions, as follows: Figure 6 As shown, incubation in PBS buffer and culture medium (DMEM, containing 10% serum) at pH 7.4 did not significantly alter the particle size of ALA-Lipo, demonstrating the stability of this lipid assembly in normal physiological environments. Furthermore, the particle size of ALA-Lipo decreased slightly in PBS buffer at pH 6.5 and pH 5.5, possibly due to the breakage of hydrazone bonds under acidic conditions, leading to the release of 5-ALA loaded on the lipid assembly surface and thus reducing the particle size, reflecting the acid-responsive nature of ALA-Lipo.

[0064] Example 5

[0065] Drug release from ALA-Lipo liposome assemblies:

[0066] 1 mL of ALA-Lipo was placed in a dialysis bag (MWCO: 1000 Da) and immersed in 40 mL of PBS buffer (pH 7.4, pH 6.5, and pH 5.5, respectively). At set time intervals, 1 mL of sample was collected from the release medium, and fresh release medium was added. 5-ALA can be quantified using a standard curve method. Specifically, an appropriate volume of 0.1 mg / mL ALA standard solution (sample solution), 4 mL of pH 5.8 sodium acetate buffer, and 4 mL of ethyl acetoacetate solution were mixed and reacted at 100 °C for 25 minutes. Then, 4 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mg / mL iron standard solution were added to the reaction mixture, and the reaction was continued at 100 °C for 30 minutes. After the reaction, the reaction mixture was allowed to cool to room temperature, and the absorbance at 480 nm was measured. A standard curve was plotted, and the concentration of 5-ALA was determined.

[0067] The release curve of 5-ALA in the ALA-Lipo lipid assembly over 48 hours is shown below. Figure 7 As shown, approximately 40% of 5-ALA is released at pH 6.5, approximately 80% at pH 5.5, and less than 20% at the normal physiological pH of 7.4. Therefore, this lipid assembly can selectively release 5-ALA via hydrazone bond cleavage in the acidic environment of a tumor.

[0068] Example 6

[0069] Radiochemical labeling of ALA-Lipo and labeling stability:

[0070] 89 Zr labeling: [The following text appears to be a separate, unrelated section:] Using HEPES buffer... 89 The pH of the Zr(Ox)2 solution was adjusted to approximately 7.0, and then mixed with 1 mL of ALA-Lipo lipid assembly solution. The mixture was incubated at 37°C for 30 minutes, and unlabeled components were removed by ultrafiltration and centrifugation. 89 Zr. Then take an appropriate amount 89 Zr-labeled ALA-Lipo solution ( 89 Zr-ALA-Lipo (radioactive dose denoted as D1) is mixed with 1 mL of PBS solution or PBS solution containing 10% FBS. The mixture is added to an ultrafiltration tube (3000 Da MWCO) and incubated at 37°C. At the set time points, the mixture is centrifuged at 4500 rpm for 10 minutes. The radioactivity of the supernatant is measured (radioactive dose denoted as D2), and the labeling rate is calculated according to the following formula:

[0071] Labeling rate (%) = (D1-D2) / D1 × 100%;

[0072] 177 Labeling of Lu: Use a 0.2 mol / L ammonium acetate solution to label the product containing Lu. 177 The pH of the LuCl3 solution was adjusted to approximately 6, and then mixed with 1 mL of ALA-Lipo lipid assembly solution. The mixture was then incubated at 37°C for 30 minutes with shaking. After the reaction was complete, an appropriate amount of... 177 Lu-labeled ALA-Lipo solution ( 177 Lu-ALA-Lipo) was mixed with 1 mL of PBS solution or PBS solution containing 10% FBS. The mixture was added to an ultrafiltration tube (3000 Da MWCO) and incubated at 37°C. The labeling rate and stability were determined in the same manner. 89 The Zr marking;

[0073] 68 Labeling of Ga: Use a 1 mol / L NaOH solution to label the contents of Ga. 68 The pH of the Ga radioactive solution was adjusted to approximately 5.5, and then mixed with 1 mL of ALA-Lipo lipid assembly solution. The mixture was then incubated at 37°C for 5 minutes. After the reaction was complete, an appropriate amount of... 68 Ga-labeled ALA-Lipo solution ( 68 Ga-ALA-Lipo) was mixed with 1 mL of PBS solution or PBS solution containing 10% FBS. The mixture was added to an ultrafiltration tube (3000 Da MWCO) and incubated at 37°C. The labeling rate and stability were determined in the same manner. 89 The Zr marking;

[0074] 64 Cu labeling: Use a 0.5 mol / L hydrochloric acid solution to label the contents of Cu. 64 The pH of the radioactive Cu solution was adjusted to approximately 5.0, and then mixed with 1 mL of ALA-Lipo lipid assembly solution. The mixture was then incubated at 37°C for 30 minutes. After the reaction was complete, an appropriate amount of... 64 Cu-labeled ALA-Lipo solution ( 64 Cu-ALA-Lipo) was mixed with 1 mL of PBS solution or PBS solution containing 10% FBS. The mixture was added to an ultrafiltration tube (3000 Da MWCO) and incubated at 37°C. The labeling rate and stability were determined in the same manner. 89 The Zr label.

[0075] 89 The labeling stability of Zr-ALA-Lipo in PBS buffer and PBS buffer containing 10% FBS is as follows: Figure 8 As shown. Among them 89The labeling efficiency of Zr-ALA-Lipo was approximately 99%, and it exhibited good labeling stability in both buffer systems within 24 hours, with no label delamination.

[0076] Example 7

[0077] ALA-Lipo lipid assemblies and PpIX in 89 Singlet oxygen production under Zr excitation:

[0078] Singlet oxygen production in ALA-Lipo lipid assemblies and PpIX was determined by measuring fluorescence changes of the singlet oxygen probe SOSG. In short, different radioactive activities at pH 5.5 were used... 89 Zr-ALA-Lipo lipid assembly solution and PpIX+ 89 The Zr mixed solution was mixed with SOSG solution (5 μM, 2 mL in a cuvette) and reacted at room temperature for 1 min. The measurement was performed under light-protected conditions. After the reaction, the fluorescence intensity was measured using a fluorescence spectrophotometer (PerkinElmer, FL6500), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0079] ALA-Lipo lipid assemblies and PpIX in 89 Zr-excited singlet oxygen production, such as Figure 9 As shown in the figure, compared with the SOSG probe solution alone, the ALA-Lipo lipid assemblies at different activities under pH 5.5 conditions... 89 Zr excitation produces virtually no singlet oxygen; the slight increase in fluorescence may be due to the effect of pH on the SOSG probe. This is because, in the absence of the relevant synthase in the in vitro environment, 5-ALA released from the ALA-Lipo lipid assembly under acidic conditions cannot be converted to PpIX, thus lacking photodynamic effect. To further verify whether the PpIX generated from 5-ALA conversion can be... 89 Zr excitation produces photodynamic effects, and we also measured the effects at different activities in an in vitro solution environment. 89 Singlet yield of PpIX under Zr excitation. Figure 9 PpIX can be compared with individual SOSG probe solutions. 89 Zr excitation produces a significant amount of singlet oxygen, which indirectly confirms this. 89 Feasibility of Zr-ALA-Lipo treatment.

[0080] Example 8:

[0081] Different cell lines 89 Zr-ALA-Lipo intake levels:

[0082] Different cells were spaced at 1×10⁻⁶ per well. 6 Cells were seeded at a density of 1 μCi in 12-well plates. Lipid assemblies labeled with 1 μCi were then seeded. 89 Zr-ALA-Lipo was co-incubated with different cell lines at 37°C. At different time points, untaken uptake was washed away with PBS. 89 After Zr-ALA-Lipo, the cells were digested with trypsin and collected. Radioactivity was measured using a gamma counter, and the cell uptake rate at different time points was calculated.

[0083] Different cell lines 89 Zr-ALA-Lipo intake levels are as follows Figure 10 As shown in the figure, we compared four different cell lines (human normal hepatocytes L02, human hepatocellular carcinoma cells HepG2, mouse breast cancer cells 4T1, and mouse colon cancer cells MC38). The figure shows that at both 12h and 24h time points, the uptake by all three tumor cell lines was slightly higher than that by normal cells. Specifically, MC38 cells showed the highest uptake. 89 Zr-ALA-Lipo uptake was the highest, likely due to the significantly higher metabolic rate in tumor cells compared to normal cells. However, there was no significant difference in uptake levels among the four cell types, because... 89 Zr-ALA-Lipo lacks actively targeted groups. However 89 Zr-ALA-Lipo can accumulate in tumor tissue through the EPR effect, thereby achieving sufficient therapeutic concentrations.

[0084] Example 9:

[0085] Cytotoxicity results of ALA-Lipo on MC 38 cells at different radionuclide doses:

[0086] The internationally recognized MTT assay was used to analyze the toxicity of the materials. MC38 cells were used as the test subject. The ALA-Lipo lipid assembly was diluted by a certain factor to achieve a final 5-ALA concentration of 86 μM, and different doses of [unspecified ingredient] were added. 89 Zr was used to co-culture cells with a blank control and replicates for 24 hours.

[0087] Add 20 μL of pre-prepared MTT solution (5 mg / mL) to each well and incubate for 4 h. Carefully remove the MTT solution, add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake thoroughly for 10 min, and measure the absorbance (OD) at 570 nm using a microplate reader. Use the average OD value of the three replicates as the OD value of the target sample and calculate the cell viability.

[0088] Cell viability = (sample OD / blank control group OD) × 100%

[0089] The cytotoxicity results of ALA-Lipo on MC 38 cells at different radionuclide doses are as follows: Figure 11 As shown, different concentrations of ALA-Lipo alone, when co-incubated with cells, exhibited low cytotoxicity, demonstrating the good biocompatibility of the ALA-Lipo lipid assembly material. Low doses... 89 Zr (1 μCi and 10 μCi) showed virtually no cytotoxicity when incubated with cells alone, but high doses... 89 When Zr (20 μCi) was incubated alone with cells, cell viability was slightly reduced due to radiation. ALA-Lipo and... 89 When Zr is used in combination, although a dose of 20 μCi is more toxic, considering the radiation damage of high-dose radionuclides to normal tissues, a dose of 10 μCi is a better choice. 89 When Zr is used in combination with ALA-Lipo, cell viability can also be reduced to about 30%.

[0090] Example 10:

[0091] ROS production in MC38 cells under different drug administration groups:

[0092] The test subjects were MC38 cells. ALA-Lipo, 89 Zr-Lipo, 89 Zr-ALA-Lipo were diluted at certain ratios to achieve a final concentration of 86 mM for 5-ALA. A blank control group and replicates were set up and incubated in an incubator for 6 h. After washing the cells with PBS, fresh DMEM medium containing DCFH-DA (10 mM) was added to each well. The cells were incubated at 37°C for 20 min, washed again with PBS, and observed under a fluorescence microscope.

[0093] The fluorescence patterns of different groups after incubation for 6 hours are shown below. Figure 12 As shown, 89 The Zr-ALA-Lipo group showed more pronounced green fluorescence compared to other groups, indicating that... 89 Zr-ALA-Lipo can generate more ROS within cells, thereby killing cancer cells.

[0094] Example 11:

[0095] Calcein-AM and PI double staining results in different drug administration groups:

[0096] The test subjects were MC38 cells. ALA-Lipo, 89 Zr-Lipo, 89Zr-ALA-Lipo were diluted at specific ratios to achieve a final concentration of 86 mM for 5-ALA. Blank groups and replicates were set up and incubated in an incubator for 6 h. After washing the cells with PBS, the cells were stained with Calcein AM / PI kit and the cells were imaged using a fluorescence microscope.

[0097] After 6 hours of incubation with different groups, the Calcein-AM / PI staining fluorescence patterns are shown below. Figure 13 As shown, 89 The Zr-ALA-Lipo group showed more pronounced red fluorescence compared to other groups, indicating that... 89 Zr-ALA-Lipo can induce more cell apoptosis, resulting in better therapeutic effects.

[0098] Example 12

[0099] In vivo antitumor effects of FA-Hemesome-ART nanovesicles and their control group:

[0100] (1) Establishment of MC38 cell-C57BL / 6 mouse model tumor-bearing mouse model: 1×10 6 MC38 cells were subcutaneously injected into C57BL / 6 mice. Tumors grew to 60 mm in the mice within 5-7 days. 3 Treatment begins on the left and right sides;

[0101] (2) The drug administration groups were set as follows: PBS, ALA-Lipo, 89 Zr-Lipo, 89 Zr-ALA-Lipo( 89 Zr dose was 400 μCi, 5-ALA dose was 5 mM; after intravenous administration, the body weight and tumor size of the mice were weighed and measured every two days. The tumor size was calculated using the following formula:

[0102] V = length × width × width / 2.

[0103] Tumor growth curves in mice after different treatment groups are as follows: Figure 14 As shown, compared to other control groups 89 Zr-ALA-Lipo showed remarkable therapeutic effects, with the tumor completely eliminated by day 6, while in other treatment groups, the tumors grew to about 20 times their initial size by day 14 after treatment.

[0104] if Figure 15As shown, the mice did not show a significant trend of weight loss, indicating that the material did not cause significant damage to the mice. This invention uses lipid assemblies as carriers and Cherenkov radiation to achieve photodynamic therapy (PDT) of deep tumors, while minimizing toxic side effects on normal tissues during the treatment process. On the one hand, by exciting the photosensitizer with Cherenkov radiation, the problem of tissue penetration of the light source can be ignored, enabling PDT treatment of deep tumors; on the other hand, the 5-ALA prodrug properties and the hydrazone-sensitive dual-lock design can minimize toxic side effects on normal tissues during treatment. Furthermore, using liposomes, a biocompatible carrier, can effectively improve the biodistribution and pharmacokinetics of 5-ALA, enhancing efficacy. In addition, liposomes can also be used to load some effective chemotherapeutic drugs, further achieving the goal of combined tumor treatment.

Claims

1. A lipid assembly loaded with 5-aminolevulinic acid, characterized in that, The lipid assembly comprises a phospholipid-chelating agent conjugate formed by a chelation reaction between a reactive phospholipid PEG reagent and a metal chelating agent. The lipid assembly is a hollow sphere with a hydrophobic bilayer on the outer shell and a hydrophilic bilayer on the inner shell. The lipid assembly also includes 5-aminolevulinic acid coupled to the hydrophobic bilayer via hydrazone bonds. The phospholipid-chelating agent conjugate is radiolabeled with a zirconium-89 radionuclide. The reactive phospholipid PEG reagent is DSPE-PEG2000-NH2, where -Ch represents the metal chelating agent. The structural formula of the phospholipid-chelating agent conjugate is shown below. 。 2. The lipid assembly according to claim 1, characterized in that, The metal chelating agent is one of deferoxamine, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-N,N',N''-triacetic acid, 1,4,8,11-tetra(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane, 1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetraazacyclotetradecane, 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15) or 11,13-triene-3,6,9-triacetic acid.

3. The method for preparing the lipid assembly according to claim 1, characterized in that, Includes the following steps: (1) Dissolve the reactive phospholipid PEG reagent in dimethyl sulfoxide, then add a metal chelating agent, and after the reaction is completed, dialyze and freeze dry to obtain the phospholipid-chelating agent conjugate; (2) Lyso PC phospholipid was dissolved in anhydrous dichloromethane solvent, and triethylamine and triphosgene were added dropwise to react and evaporate to obtain intermediate Lipid-COCl; then Lipid-COCl was added dropwise to a dichloromethane solution of NHNH2·H2O, and the reaction was washed and centrifuged to obtain Lipid-NHNH2 conjugate; finally, an ethanol solution of aminolevulinic acid hydrochloride was added dropwise, glacial acetic acid was added dropwise to react in the dark, n-hexane was added, and hydrazone coupling was obtained to obtain Lipid-ALA conjugate; (3) Dispalmitoylphosphatidylcholine, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate and Lipid-ALA conjugate are dissolved in an organic solvent, and lipid assembly ALA-Lipo is obtained by film hydration method and extrusion. (4) The lipid assembly ALA-Lipo was mixed with a radioactive nuclide solution for labeling to obtain a radioactive lipid assembly loaded with 5-aminolevulinic acid.

4. The preparation method according to claim 3, characterized in that, In step (1), the mass ratio of the reactive phospholipid PEG reagent to the metal chelating agent is 5:1~3.

5. The preparation method according to claim 3, characterized in that, In step (2), the mass ratio of the phospholipid Lyso PC, triethylamine and triphosgene is 50:0.012~0.019:150~300, the mass ratio of Lipid-COCl to NHNH2·H2O is 50:44~65, and the mass ratio of Lipid-NHNH2 conjugate to aminolevulinic acid hydrochloride is 50:15~30.

6. The preparation method according to claim 3, characterized in that, The specific synthesis route for step (2) is as follows: 。 7. The preparation method according to claim 3, characterized in that, The molar ratio of dipalmitoylphosphatidylcholine, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000, phospholipid-chelating agent conjugate, and Lipid-ALA conjugate is 41~42:26~27:1.2:2.0:28~29.

8. The preparation method according to claim 3, characterized in that, In step (4), 1 mL of the lipid assembly ALA-Lipo reacts with and is labeled with 0.1-1 mCi of radionuclide solution.

9. The use of the lipid assembly according to claim 1 in the preparation of targeted drugs, characterized in that, The outer hydrophobic bilayer of the lipid assembly shell is loaded with hydrophobic chemotherapeutic drugs and / or the inner hydrophilic bilayer is loaded with water-soluble chemotherapeutic drugs.

10. The application according to claim 9, characterized in that, The mass ratio of the hydrophobic or water-soluble chemotherapeutic drug to the liposome assembly is 5-10:100.

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