PSMA receptor-targeted nano-composite, preparation method and application of PSMA receptor-targeted nano-composite

Abstract

The invention discloses a PSMA receptor-targeted nano-composite as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The nanocomposite can be combined with a PSMA receptor with high specificity, so that PSMA positive cells can be accurately targeted. The contrast agent carried by the liposome has phase change capability, and can be changed into a gaseous state from a liquid state under the action of heating or ultrasound and laser, so that the targeted part of the liposome is imaged. The photothermal probe or the fluorescent molecule of the liposome shell can emit a fluorescent signal with high sensitivity, so that the nano-composite can perform fluorescence-ultrasonic-photoacoustic imaging, has the advantages of high sensitivity, high specificity and high spatial resolution, and can detect prostate cancer in an early stage and monitor recurrence and metastasis of the prostate cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials. Specifically, the present invention relates to a nano - complex targeting PSMA receptor, a preparation method thereof, and an application thereof. Background Art

[0002] Since the accurate imaging of biological targets is an important tool for understanding biological phenomena or diagnosing various diseases without error, currently, multimodal imaging is gradually becoming an important means. By combining dual and triple modalities, many disadvantages of single - modality imaging can be overcome. For example, for the early diagnosis of cancer, techniques that simultaneously use positron emission tomography (PET) allowing functional imaging with high sensitivity and computed tomography (CT) capable of providing clear anatomical information have been proposed. Imaging techniques using different modalities can be combined. For example, the combination of magnetic resonance (MR) / optical and photoacoustic tomography (PAT) / ultrasound can be achieved.

[0003] Currently, most of the targeting diagnostic probes for prostate cancer are radionuclides, lacking ultrasound, photoacoustic, and fluorescence multimodal imaging probes. Radionuclide probes limit the frequency of repeated examinations and require particular caution when used in young patients. In addition, radionuclide probes cannot provide real - time dynamic information such as tumor angiogenesis and metabolic microenvironment. In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a nano - complex targeting PSMA receptor, a preparation method thereof, and an application thereof, so as to provide imaging technical support for the early detection, recurrence monitoring, metastasis monitoring, etc. of prostate cancer, and help to guide treatment.

[0005] The present invention is implemented as follows:

[0006] In the first aspect, the present invention provides a nano - complex targeting PSMA receptor. The nano - complex has a core - shell structure, and the outer shell is a liposome. The liposome includes: lipids, lipid - molecule - PSMA - targeting - peptide covalent conjugates, lipid - molecule - active - molecule covalent conjugates, and lipid - molecule - hydrophilic - segment.

[0007] The inner core includes a contrast agent; the contrast agent is a material that undergoes a phase change under ultrasonic, heating, or laser conditions. The amino acid sequence of the PSMA - targeting peptide is shown as any one of SEQ ID NO: 1 - 4 or the amino acid sequence is (WQPDTAHHWATL)2 - K. The active molecule is selected from a photo - thermal probe or a fluorescent molecule.

[0008] In the second aspect, the present invention also provides a preparation method of a nano - complex targeting PSMA receptor, which includes the following steps:

[0009] Covalently conjugate a lipid molecule-PSMA targeting peptide conjugate, a lipid molecule-active molecule conjugate, lipids and a lipid molecule-hydrophilic segment, self-assemble to form liposomes, and then mix a contrast agent with the liposomes and perform sonication to obtain a nano-complex.

[0010] In a third aspect, the present invention also provides the use of a nano-complex targeting the PSMA receptor or a nano-complex targeting the PSMA receptor prepared by the above preparation method in the preparation of products for the diagnosis, recurrence monitoring or cancer cell metastasis monitoring of prostate cancer.

[0011] The present invention has the following beneficial effects:

[0012] The present invention provides a nano-complex for use in the diagnosis, recurrence monitoring or cancer cell metastasis monitoring of prostate cancer. The liposomes of this nano-complex carry a PSMA targeting peptide and can specifically bind to the PSMA receptor with high specificity, thereby precisely targeting PSMA-positive cells. The contrast agent carried by the liposomes has the ability to change phase and can change from a liquid state to a gaseous state under the action of heating, ultrasound or laser, so as to image the site targeted by the liposomes. The photothermal probe or fluorescent molecule carried by the liposome shell can emit fluorescent signals with high sensitivity. Therefore, this nano-complex can perform fluorescence-ultrasound-photoacoustic imaging, has the advantages of high sensitivity, high specificity and high spatial resolution, and can detect prostate cancer at an early stage and monitor its recurrence and metastasis to guide treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 Results of the preparation and characterization of nanoparticles (A FTIR results of DSPE-Peg-CY7.5, B FTIR results of DSPE-Peg-pep, C surface plasmon resonance detection of the affinity of pep for PSMA, D transmission electron microscopy observation of particle morphology (scale bar 200 nm), E particle size analyzer detection of particle size and distribution, F particle potential detection);

[0015] Figure 2Experimental result graphs showing the high specificity of nanoparticles for PSMA-positive prostate cancer cells in vitro; fluorescence microscopy images of nanoparticles binding to C4-2 (A), DU145 tumors, LNCaP tumors (C), and PC3 tumors (D) respectively, E is the semi-quantitative result of the fluorescence intensity in Figures A - B, and F is the semi-quantitative result of the fluorescence intensity in Figures C - D;

[0016] Figure 3 In vivo imaging results of four groups of tumor-bearing mice after injection of nanoparticles, A shows in vivo imaging, and B shows ex vivo tissue and tumor imaging;

[0017] Figure 4 Results of two-dimensional and contrast-enhanced ultrasound imaging; A shows in vitro imaging of nanoparticles, and B shows in vivo tumor imaging;

[0018] Figure 5 Results of photoacoustic imaging; A shows the detection of the photothermal effect of the particles, B shows the in vitro photoacoustic curve of the particles, C shows the photoacoustic imaging results of four groups of tumor-bearing mice after injection of the particles, and D is the statistical result of Figure C. Detailed implementation manners

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0020] Glossary:

[0021] PSMA, prostate-specific antigen.

[0022] Liposomes are formed by amphiphilic compounds containing phospholipids. Usually, the amphiphilic compounds are arranged at the interface between a substantially water-insoluble organic solvent and an aqueous medium to stabilize the emulsified solvent microbubbles. Amphiphilic compounds include compounds containing molecules with a hydrophilic polar head (e.g., polar or ionic group) capable of reacting with the aqueous medium, and, for example, a hydrophobic organic tail (e.g., hydrocarbon chain) capable of reacting with the organic solvent. Amphiphilic compounds are compounds that can stabilize mixtures of substances that cannot usually be mixed by other methods, such as mixtures of two immiscible liquids (e.g., water and oil), mixtures of a liquid and a gas (e.g., gas microbubbles in water), or mixtures of a liquid and insoluble particles (e.g., metal nanoparticles in water).

[0023] Amphiphilic phospholipid compounds contain at least one phosphate group and at least one, preferably two, lipophilic long-chain hydrocarbon groups.

[0024] As amphiphilic phospholipids, known compounds can be used. Examples thereof include diphytanoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-bis-O-dodecyl-sn-glycero-3-phosphocholine (DIODPC) (1,2-DiODodecyl-sn-glycero-3-phosphocholine), dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol, dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-[phosphatidyl-rac-(1-glycerol)] (DMPG), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso PC), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (Lyso PE), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt) [1,2-dierucoyl-sn-glycero-3-phosphate(sodium salt)(DEPA-NA)], 1,2-erucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-linoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt) (DLPA-NA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt) (DLPS-NA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA-NA), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt) (DMPS-NA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA-NA), 1,2-oleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt) (DOPS-NA), 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA-NA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt) (DPPS-NA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA-NA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2-diostearpyl-sn-glycero-3-phosphoethanolamine) (DSPE), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SOPC), and 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC).

[0025] In addition, as the amphiphilic phospholipid compound, a modified phospholipid compound can be used. Examples of the modified phospholipid compound include examples of the phospholipid modified with polyethylene glycol (PEG) such as phosphatidylethanolamine modified with polyethylene glycol (PEG) (DMPE-PEG) (phosphatidylethanolamine) or phosphoethanolamine modified with polyethylene glycol (PEG) (DSPE-PEG) (phosphoethanolamine).

[0026] According to an exemplary embodiment of the present invention, the amphiphilic phospholipid compound used in the present invention may include N-hydroxysuccinimide (NHS) for forming an amide bond.

[0027] In addition to the amphiphilic compound, the present invention may further include additional amphiphilic substances, examples of which include lysophospholipid, stearic acid, polyethylene glycol, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid stearate, and polyoxyethylene fatty alcohol.

[0028] In a first aspect, the present invention provides a nano - complex targeting the PSMA receptor. The nano - complex has a core - shell structure, where the outer shell is a liposome. The liposome includes: lipids, a lipid - molecule - PSMA targeting peptide covalent conjugate, a lipid - molecule - active molecule covalent conjugate, and a lipid - molecule - hydrophilic segment;

[0029] The inner core includes a contrast agent; the contrast agent is a material that undergoes a phase change under ultrasonic, heating, or laser conditions. The amino acid sequence of the PSMA targeting peptide is shown as any one of SEQ ID NO: 1 - 4 or the amino acid sequence is (WQPDTAHHWATL)2 - K. The active molecule is selected from a photothermal probe or a fluorescent molecule.

[0030] The liposome of the nano - complex carries a PSMA targeting peptide, which can specifically bind to the PSMA receptor with high specificity, thereby precisely targeting PSMA - positive cells. The contrast agent carried by the liposome has the ability to undergo a phase change, and can change from a liquid state to a gaseous state under heating, ultrasonic, or laser action, thereby imaging the site targeted by the liposome. The photothermal probe or fluorescent molecule carried by the liposome shell can emit fluorescent signals with high sensitivity. Therefore, this nano - complex can perform fluorescence - ultrasound - photoacoustic imaging, having the advantages of high sensitivity, high specificity, and high spatial resolution, and can detect prostate cancer at an early stage, monitor its recurrence and metastasis, and guide treatment.

[0031] SEQ ID NO: 1: GRFLTGGTGRLLRIS.

[0032] In other embodiments, the amino acid sequence of the PSMA targeting peptide can also be SHSFSVGSGDHSPFT (SEQ ID NO: 2), WQPDTAHHWATL (SEQ ID NO: 3), (WQPDTAHHWATL)2 - K, or GTIQPYPFSWGY (SEQ ID NO: 4).

[0033] In a preferred embodiment of the application of the present invention, the lipid molecule in the lipid - molecule - PSMA targeting peptide covalent conjugate is covalently linked to the PSMA targeting peptide through a hydrophilic segment, and the lipid molecule in the lipid - molecule - active molecule covalent conjugate is covalently linked to the active molecule through a hydrophilic segment.

[0034] In a preferred embodiment of the application of the present invention, the hydrophilic segment is selected from one or more of polyethylene glycol, poly(ethylene oxide), poly(N - (2 - hydroxypropyl) methacrylamide), polyvinylpyrrolidone, and poly(methyl acrylate phosphatidylcholine);

[0035] In a preferred embodiment of the application of the present invention, the hydrophilic segment is polyethylene glycol.

[0036] In a preferred embodiment of the application of the present invention, the molar ratio of the lipid molecule-PSMA targeting peptide conjugate in the liposome is 5-25%, and the molar ratio of the lipid molecule-fluorescent molecule conjugate in the liposome is 1-10%. The molar ratio of the lipid molecule-hydrophilic segment in the liposome is 1-5%.

[0037] For example, the molar ratio of the lipid molecule-PSMA targeting peptide conjugate in the liposome is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%. The molar ratio of the lipid molecule-fluorescent molecule conjugate in the liposome is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. Such as the molar ratio of the lipid molecule-hydrophilic segment in the liposome is 1%, 2%, 3%, 4% or 5%.

[0038] At the above-mentioned addition molar ratio (mole percentage), the prepared nano-complex has high specificity, sensitivity and spatial resolution. The nano-complex provided by the present invention has a wide range of application scenarios and can be used for intraoperative navigation and vascular infiltration assessment; it can define the tumor resection margin and trace lymph nodes.

[0039] In a preferred embodiment of the application of the present invention, the lipid molecule is one or more of distearoyl phosphatidylethanolamine, lecithin, soybean phospholipid, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, ceramide, cerebroside, ganglioside, glyceride and its derivatives.

[0040] In a preferred embodiment of the application of the present invention, the lipid molecule-PSMA targeting peptide conjugate is a distearoyl phosphatidylethanolamine-PSMA targeting peptide conjugate; the lipid molecule-fluorescent molecule conjugate is a distearoyl phosphatidylethanolamine-fluorescent molecule conjugate.

[0041] In a preferred embodiment of the application of the present invention, the molar ratio of the lipid in the liposome is 60-90%. For example, it is 60%, 65%, 70%, 72%, 75%, 80%, 85% or 90%.

[0042] At the above-mentioned mixed molar ratio, it has the technical advantages of good stability and good enrichment effect of nano-sized particles.

[0043] The PSMA targeting peptide is connected to the hydrophilic segment connected to the end of the lipid molecule by any one of the following ways: ester group, amide group, ether group, thioether and carbamate;

[0044] The hydrophilic chain segment with the active molecule connected to the end of the lipid molecule is connected by any one of the following ways: ester group, amide group, ether group, thioether, carbamate group.

[0045] In a preferred embodiment of the application of the present invention, the PSMA targeting peptide binds to the hydroxyl group of the hydrophilic chain segment with the carboxyl group connected to the end of the lipid molecule, and the active molecule binds to the amino group or hydroxyl group of the hydrophilic chain segment with the carboxyl group connected to the end of the lipid molecule.

[0046] In a preferred embodiment of the application of the present invention, the particle size of the nano - complex is 260 ± 14 nm.

[0047] In a preferred embodiment of the application of the present invention, the contrast agent is a fluorine - containing liquid;

[0048] In a preferred embodiment of the application of the present invention, the fluorine - containing liquid is selected from perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane or sulfur hexafluoride.

[0049] In a preferred embodiment of the application of the present invention, the fluorescent molecules include but are not limited to Cy3, Cy3.5, Cy5, Cy5.5, 5 - FAM, 6 - FAM, HEX, TET, VIC, JOE, Quasar670, NED, TAMRA, ROX or Texas Red;

[0050] The photothermal probes include but are not limited to small molecule compounds such as ICG, IR780, IR783, Cy7.5 and IR808.

[0051] In a second aspect, the present invention also provides a preparation method of a nano - complex targeting the PSMA receptor, which comprises the following steps:

[0052] Covalently conjugate the lipid molecule - PSMA targeting peptide, the lipid molecule - active molecule covalently conjugate, lipids and the lipid molecule - hydrophilic chain segment, self - assemble to form liposomes, and then mix the contrast agent with the liposomes and perform ultrasonic treatment to obtain the nano - complex.

[0053] In one embodiment, the liposomes are prepared by the ethanol injection method, or can also be prepared into liposomes by the thin - film hydration method. The steps of preparing the liposomes also include dialysis treatment to remove the organic phase in the liposome solution (the solution after the liposome mother liquor is injected into the aqueous phase).

[0054] After mixing the contrast agent with the liposomes, for example, the nano - complex can be obtained under ultrasonic treatment of a probe - type ultrasonic instrument (UP4000, USA) (10% power, 2 minutes).

[0055] In a third aspect, the present invention also provides the use of a nanocomplex targeting the PSMA receptor or a nanocomplex targeting the PSMA receptor prepared by the above preparation method in the preparation of products for the diagnosis, recurrence monitoring, or cancer cell metastasis monitoring of prostate cancer;

[0056] In a preferred embodiment of the application of the present invention, the products for the diagnosis, recurrence monitoring, or cancer cell metastasis monitoring of prostate cancer are used for at least one of the following imaging:

[0057] Fluorescence imaging, photoacoustic imaging, and ultrasonic imaging.

[0058] The inventors found that after the nanocomplex provided by the present invention is administered to an animal, in vivo and in vitro fluorescence imaging, ultrasonic imaging, and photothermal imaging can be performed. It is enriched at the target tumor site through the EPR effect, and after laser irradiation, it vaporizes from nanoscale size to micron-scale size, which can enhance the signals of ultrasonic imaging and photoacoustic imaging. The nanocomplex has a high affinity for PSMA-positive tumors and obtains energy under laser irradiation at, for example, 758 nm for photothermal imaging.

[0059] The nanocomplex provided by the present invention can be used for PAT or ultrasonic imaging diagnosis.

[0060] The EPR effect (Enhanced Permeability and Retention Effect) is a unique phenomenon observed in tumor tissues. Due to the high permeability of tumor blood vessels and poor lymphatic return, macromolecular substances (such as nanoparticles, liposomes, etc.) selectively accumulate and remain in tumor tissues for a long time.

[0061] The nanocomplex of the present invention is intravenously injected.

[0062] The appropriate dosage of the nanocomplex of the present invention can vary according to factors such as the formulation method, administration method, the age, weight, gender, or disease condition of the patient, diet, administration time, administration route, excretion rate, and reaction sensitivity. At the same time, the dosage of the contrast agent of the present invention for rats can preferably be 10 mg / kg (body weight).

[0063] The features and properties of the present invention are further described in detail below in conjunction with examples.

[0064] Example 1

[0065] This example provides a preparation method of a nanocomplex. The amino acid sequence of the PSMA targeting peptide (i.e., polypeptide, abbreviated as pep) in the nanocomplex is GRFLTGGTGRLLRIS. The active molecule is CY7.5, and the imaging agent is perfluorohexane (PFH).

[0066] The specific preparation method is as follows:

[0067] Step 1: Synthesize DSPE-PEG2000-pep polypeptide and DSPE-PEG-CY7.5:

[0068] First, couple CY7.5 or pep to DSPE-PEG-NHS separately to synthesize amphiphilic conjugates. DSPE-PEG-NHS reacts with CY7.5-NH2 / pep in DMF at 25 °C for 24 h, and triethylamine is added at a molar ratio of amino group:triethylamine = 1:3. The reactants are dialyzed in a dialysis bag (MWCO: 10 kDa) for 12 h to remove unreacted CY7.5-NH2 / pep and excess triethylamine. The dialysis medium is distilled water, and after changing the water twice, the sample is taken out and freeze-dried.

[0069] Step 2: Prepare liposomes: DSPE-PEG-CY7.5 and DSPE-PEG-pep are dispersed in ethanol (5 mg / mL), and a mother liquor is prepared with DSPC:DSPE-PEG-pep:DSPE-PEG-CY7.5:DSPE-PEG 2000 = 3:5:1.5:0.5 (wt). The molar ratio of DSPC:DSPE-PEG-pep:DSPE-PEG-CY7.5:DSPE-PEG 2000 is 64.38:23.29:9.4:2.93. Liposomes are prepared by the ethanol injection method. The mother liquor is slowly injected into nine times the volume of the aqueous phase in a water bath with ultrasonic treatment to obtain CP-Lipo (i.e., liposomes), and the organic phase is removed by dialysis in a dialysis bag (MWCO: 10 kDa) for 12 h.

[0070] Step 3: Mix perfluorohexane (PFH) and CP-Lipo, and obtain CP@PFH (i.e., nanocomplex) under ultrasonic treatment with a probe-type ultrasonic device (UP4000, USA) (10% power, 2 minutes).

[0071] Experimental Example 1

[0072] Analyze DSPE-PEG-CY7.5 and DSPE-PEG-pep prepared in Example 1 by FTIR (Fourier transform infrared spectrometer) respectively, and the results are shown in A and B in Figure 1 . The FTIR results show that DSPE-PEG-CY7.5 and DSPE-PEG-pep are successfully synthesized.

[0073] Detect the affinity of pep for PSMA by surface plasmon resonance, and the results are shown in C in Figure 1 . Pep has a high specific binding ability to the prostate-specific membrane antigen (PSMA) receptor, and its affinity detected by surface plasmon resonance is (7.627 ± 3.4)E-7 M.

[0074] The morphology of CP@PFH particles was observed by transmission electron microscopy (scale bar: 200 nm), and the particles were found to be nearly circular in shape with a size of approximately 200 nm ( Figure 1 D in Figure 1 ). The particle size was measured by a particle size analyzer to be 260 ± 14 nm with a uniform particle size distribution ( Figure 1 E in

[0075] Experimental Example 2

[0076] The targeting ability of CP@PFH particles to prostate cancer was detected in vitro.

[0077] Four types of cells were cultured on 24-well cell culture slides pre-coated with Poly-L-Lysine (0.1 mg / ml) (5×10 5 cells) for 24 hours, and then the medium was replaced with 0.5 mL of three fresh media containing modified targeting peptide CP@PFH, unmodified targeting peptide C@PFH, and no addition (control). After incubating the cells for another 4 hours, the medium was removed and the cells were washed with PBS. After fixation, the cells were mounted with a mounting medium containing DAPI stain, and the cells were imaged by confocal laser scanning microscopy.

[0078] The particles showed high selectivity for C4-2 and LNCaP tumor cells (PSMA-positive), and the fluorescence intensity was 8.85 - 12.86 times that of DU145 and PC3 tumor cells (PSMA-negative cells), referring to Figure 2 A - F in

[0079] Experimental Example 3

[0080] Fluorescence imaging of the CP@PFH particles prepared in Example 1 was performed.

[0081] For in vivo imaging, tumor-bearing mice with tumors growing to 100 mm 3 were used. After anesthesia, 10 mg / kg of the particles were injected via the tail vein. The nude mice were from Nude, and the four types of tumors were: PSMA-positive C4-2 and LncaP, and PSMA-negative DU145 and PC3.

[0082] As shown in Figure 3 , the in vivo imaging results ( Figure 3 A in

[0083] Experimental Example 4

[0084] Ultrasonic imaging was performed on the CP@PFH particles prepared in Example 1. When the tumor volume reached approximately 100 mm 3 , after intraperitoneal anesthesia of the mice, the particles (10 mg / kg) were intravenously injected into the tumor-bearing mice. Contrast examination was performed using an ultrasonic imaging system VINNO70 (mechanical index 0.2, probe frequency 18 MHz) to observe the circulation of microbubbles in the tumor region.

[0085] Figure 4 Shown is the detection of the ultrasonic imaging ability of the particles in vitro and in vivo. Figure 4 As shown in A, after 12 h of intravenous injection of the particles, laser irradiation was performed on them (CP@PFH&Laser). After laser irradiation, the particles obtained energy and vaporized, and they had the ability of ultrasonic imaging. Figure 4 B in it is the in vivo imaging effect, showing the high affinity of the particles for PSMA-positive tumors.

[0086] Experimental Example 5

[0087] Photoacoustic imaging was performed on the CP@PFH particles prepared in Example 1.

[0088] When the tumor volume reached approximately 100 mm 3 , the particles were intravenously injected into the tumor-bearing mice for photoacoustic imaging examination. The mice were placed in a fixing table in the prone position. Excitation was selected at a wavelength of 750 nm, and the laser parameters were set to continuously collect the photoacoustic signals of the tumor at different time points. The Vevo system software was used to select the ROI for subsequent data analysis.

[0089] Figure 5 A in it is the photothermal curve of the particles, indicating that the particles obtained energy under 750-nm laser irradiation. Figure 5 B in it is the in vitro photoacoustic curve of the particles, indicating that the particles have the ability of photoacoustic imaging. Figure 5 C in it is the photoacoustic imaging results of four groups of tumor-bearing mice. Figure 5 D in it is the statistical result of Figure C, and the particles have a high affinity for PSMA tumors.

[0090] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nano - complex targeting the PSMA receptor, characterized in that, The nano - composite has a core - shell structure, and the outer shell is a liposome. The liposome includes: lipids, lipid - PSMA targeting peptide covalent conjugates, lipid - active molecule covalent conjugates, and lipid - hydrophilic segment; The core includes a contrast agent; the contrast agent is a material that undergoes a phase change under ultrasonic, heating, or laser conditions. The amino acid sequence of the PSMA targeting peptide is shown as any one of SEQ ID NO: 1 - 4 or the amino acid sequence is (WQPDTAHHWATL)2 - K; the active molecule is selected from a photothermal probe or a fluorescent molecule.

2. The nano - complex targeting the PSMA receptor according to claim 1, characterized in that, The lipid molecule in the lipid - PSMA targeting peptide covalent conjugate is covalently linked to the PSMA targeting peptide through a hydrophilic segment, and the lipid molecule in the lipid - active molecule covalent conjugate is covalently linked to the active molecule through a hydrophilic segment; Preferably, the hydrophilic segment is selected from one or more of polyethylene glycol, poly(ethylene oxide), poly(N - (2 - hydroxypropyl) methacrylamide), polyvinylpyrrolidone, and methyl acrylate phosphatidylcholine; Preferably, the hydrophilic segment is polyethylene glycol.

3. The nano - complex targeting the PSMA receptor according to claim 2, characterized in that, The molar ratio of the lipid - PSMA targeting peptide covalent conjugate in the liposome is 5 - 25%, and the molar ratio of the lipid - fluorescent molecule covalent conjugate in the liposome is 1 - 10%; the molar ratio of the lipid - hydrophilic segment in the liposome is 1 - 5%; Preferably, the lipid molecule is one or more of distearoyl phosphatidylethanolamine, lecithin, soybean phospholipid, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, ceramide, cerebroside, ganglioside, glyceride, and its derivatives; Preferably, the lipid - PSMA targeting peptide covalent conjugate is a distearoyl phosphatidylethanolamine - PSMA targeting peptide covalent conjugate; the lipid - fluorescent molecule covalent conjugate is a distearoyl phosphatidylethanolamine - fluorescent molecule covalent conjugate.

4. The nano - complex targeting the PSMA receptor according to claim 3, wherein, The molar ratio of the lipid in the liposome is 60 - 90%.

5. The nano - complex targeting the PSMA receptor according to claim 4, characterized in that, The PSMA targeting peptide is connected to the hydrophilic segment connected to the end of the lipid molecule by any one of the following ways: ester group, amide group, ether group, thioether, and carbamate; The active molecule is connected to the hydrophilic segment connected to the end of the lipid molecule by any one of the following ways: ester group, amide group, ether group, thioether, carbamate; Preferably, the PSMA targeting peptide binds to the hydroxyl group of the hydrophilic segment connected to the end of the lipid molecule through a carboxyl group, and the active molecule binds to the amino or hydroxyl group of the hydrophilic segment connected to the end of the lipid molecule through a carboxyl group.

6. The nano - complex targeting the PSMA receptor according to claim 1, characterized in that, The particle size of the nano - composite is 260 ± 14 nm.

7. The nano - complex targeting the PSMA receptor according to claim 1, characterized in that, The contrast agent is a fluorine - containing liquid; Preferably, the fluorine - containing liquid is selected from perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or sulfur hexafluoride.

8. The nano - complex targeting the PSMA receptor according to claim 1, characterized in that, The fluorescent molecule is selected from Cy3, Cy3.5, Cy5, Cy5.5, 5 - FAM, 6 - FAM, HEX, TET, VIC, JOE, Quasar670, NED, TAMRA, ROX, or Texas Red; The photothermal probe is selected from ICG, IR780, IR783, Cy7.5 or IR808.

9. A method for preparing a nano - complex targeting the PSMA receptor according to any one of claims 1 - 8, characterized in that, It comprises the following steps: Covalently conjugating a lipid molecule-PSMA targeting peptide conjugate, a lipid molecule-active molecule conjugate, lipids and a lipid molecule-hydrophilic segment, self-assembling to form liposomes, and then mixing a contrast agent with the liposomes and subjecting them to ultrasonic treatment to obtain a nano-complex.

10. Use of the nano-complex targeting the PSMA receptor according to any one of claims 1-8 or the nano-complex targeting the PSMA receptor prepared by the preparation method according to claim 9 in the preparation of products for the diagnosis, recurrence monitoring or cancer cell metastasis monitoring of prostate cancer; Preferably, the products for the diagnosis, recurrence monitoring or cancer cell metastasis monitoring of prostate cancer are used for at least one of the following imaging: Fluorescence imaging, photoacoustic imaging and ultrasonic imaging.

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