A biomimetic nanoparticle selectively targeting psma and preparation method and application thereof

Abstract

The application discloses a kind of selectively targeted PSMA's biomimetic nanoparticles and its preparation method and application.This application simulates the glutamic acid substrate of carboxypeptidase and designs new functional monomer N-acrylamido glutaric acid and N-acrylamido succinic acid, a series of targeted PSMA's biomimetic nanoparticles with high affinity and selectivity to PSMA are synthesized using new monomer.The nanoparticle has low cost, higher stability, longer service life and stronger ability to resist harsh environment, overcomes the shortcomings of high cost, low preparation efficiency, long screening cycle, difficult to save and other defects of protein antibody.This application synthesizes water-soluble carbon dots with near-infrared fluorescence and photothermal properties, combined with the targeted PSMA's biomimetic nanoparticles, can replace protein antibody, nuclide imaging molecule and be used for targeted recognition of tumor cells and effective fluorescence imaging to early diagnose cancer, further kill tumor cells through photothermal therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a kind of biomimetic nanoparticles selectively targeting PSMA and its preparation method and application. BACKGROUND

[0002] Prostate cancer is the second most common cancer in men, and the third most common cause of cancer death in men. Prostate-specific membrane antigen (PSMA) is overexpressed in 90% of metastatic prostate cancer, while the physiological expression level in normal tissues is low (prostate, small intestine, salivary gland and lacrimal gland, and kidney). The biological characteristics of PSMA specifically expressed on the surface of prostate cancer cells make it have high research value in the field of molecular imaging and targeted therapy of prostate cancer. Studies have shown that radionuclides and other substances and labeled PSMA monoclonal antibodies show certain clinical application prospects in the molecular imaging diagnosis and targeted therapy of prostate cancer. However, PSMA monoclonal antibodies belong to macromolecular protein substances, and have poor prostate cancer tumor substance penetration, long circulation half-life, large side effects in vivo, and are prone to cause immune response reactions. In addition, the production cost of monoclonal antibodies is high, and the biological activity is easy to change during storage, thus limiting their application in the diagnosis and treatment of prostate cancer. In view of this phenomenon, it is urgent to find a kind of biomolecule that can combine diagnosis and treatment drugs in one. SUMMARY

[0003] The present application aims to overcome the many defects of the detection and treatment technology of radionuclides and other substances and labeled PSMA monoclonal antibodies, and to provide a kind of biomimetic nanoparticles selectively targeting PSMA and its preparation method and application.

[0004] A preparation method of near-infrared fluorescent water-soluble carbon dots is as follows: mixing Nile blue, ethylenediaminetetraacetic acid disodium salt and water and adding them into a hydrothermal reaction kettle, reacting at 160-200 ℃ for 5-12 h, filtering the supernatant, purifying the filtrate by dialysis, and freeze-drying to obtain black powder near-infrared fluorescent water-soluble carbon dots.

[0005] The molar ratio of Nile blue to ethylenediaminetetraacetic acid disodium is 0.3-3.

[0006] The preparation method of the biomimetic nanoparticles selectively targeting PSMA is as follows: preparing a mixed aqueous solution of N-isopropyl acrylamide, N-tert-butyl acrylamide, N-acrylamidoglutaric acid, N-acrylamidobutandioic acid and N,N'-methylenebisacrylamide with a molar ratio of 40-50:30-40:5-15:5-15:2, adding a load, ultrasonic mixing, adding an initiator after removing air by nitrogen, stirring and sealing at 50-80 ℃ for 2-6 h, and finally dialyzing with water to obtain the biomimetic nanoparticles selectively targeting PSMA.

[0007] The initiator is ammonium persulfate, azobisisobutyronitrile or tetramethyl ethylenediamine.

[0008] The N The synthesis method of acrylamidoglutamic acid (AAGA) is as follows: the pH value of an aqueous amino glutaric acid solution is adjusted to 12.5-13.5 by sodium hydroxide or potassium hydroxide, then it is added dropwise into an acryloyl chloride solution under vigorous stirring and ice bath, and stirred at room temperature for 2-12 h, then the pH value is adjusted to 10-12 by sodium hydroxide or potassium hydroxide, and the organic phase is removed by washing with ethyl acetate; then the pH value is adjusted to 0-2 by hydrochloric acid, and the aqueous phase is extracted with ethyl acetate to obtain an extract, which is dried with magnesium sulfate, filtered, and precipitated in n-hexane to obtain N-acrylamidoglutamic acid.

[0009] The synthesis method of N-acrylamidoglutamic acid (AAGA) is as follows: the pH value of an aqueous amino glutaric acid solution is adjusted to 12.5-13.5 by sodium hydroxide or potassium hydroxide, then it is added dropwise into an acryloyl chloride solution under vigorous stirring and ice bath, and stirred at room temperature for 2-12 h, then the pH value is adjusted to 10-12 by sodium hydroxide or potassium hydroxide, and the organic phase is removed by washing with ethyl acetate; then the pH value is adjusted to 0-2 by hydrochloric acid, and the aqueous phase is extracted with ethyl acetate to obtain an extract, which is dried with magnesium sulfate, filtered, and precipitated in n-hexane to obtain N-acrylamidoglutamic acid.

[0010] The solvent in the acryloyl chloride solution is one or more of ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide.

[0011] The load is one or more of near-infrared fluorescent water-soluble carbon dots, fluorescein isothiocyanate, rhodamine B, paclitaxel, doxorubicin, doxorubicin hydrochloride and ferroferric oxide magnetic particles.

[0012] The application of the above-prepared selective PSMA-targeting biomimetic nanoparticles in preparing a prostate fluorescent imaging agent, a prostate cancer photothermal therapeutic agent or a photothermal antibacterial agent.

[0013] The application designs new functional monomer molecules N-acrylamidoglutamic acid and N-acrylamidoglutamic acid based on the design of substrate analogs with carboxypeptidase activity of PSMA, and synthesizes PSMA-targeting biomimetic nanoparticles with high affinity and selectivity to a series of PSMA by using the new monomers, and the affinity constant reaches 10 -9M. The biomimetic nanoparticles have low cost, high stability, long service life and strong ability to resist harsh environment, overcoming the shortcomings of high cost, low preparation efficiency, long screening period, easy variability and difficult preservation of protein antibodies. In classical visible light fluorescence imaging, since the excitation light and the emission light are located in the wavelength range of 400-650 nm, the light absorption and scattering effect of biological tissues in this wavelength range and the interference of spontaneous fluorescence seriously affect the effect of in vivo imaging. The water-soluble carbon dots with near-infrared fluorescence and photothermal properties synthesized in the application can replace protein antibodies, nuclide imaging molecules and the like to be used for targeted recognition of tumor cells and effective fluorescence imaging to early diagnose cancer, and further kill tumor cells through photothermal therapy. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a scanning electron microscope image of the near-infrared fluorescent water-soluble carbon dots in Example 1.

[0015] Figure 2 is a transmission electron microscope image of the near-infrared fluorescent water-soluble carbon dots in Example 1.

[0016] Figure 3 is a fluorescence spectrum of the near-infrared fluorescent water-soluble carbon dots and the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0017] Figure 4 is a scanning electron microscope image of the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0018] Figure 5 is a transmission electron microscope image of the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0019] Figure 6 is a binding isotherm curve of the biomimetic nanoparticles selectively targeting PSMA, PSMA polypeptide, PSMA protein and impurity protein in Example 1.

[0020] Figure 7 is a temperature change graph of the biomimetic nanoparticles selectively targeting PSMA in Example 1 under laser irradiation at different concentrations.

[0021] Figure 8 is a temperature change graph of the biomimetic nanoparticles selectively targeting PSMA in Example 1 under laser irradiation at different laser powers.

[0022] Figure 9 is a temperature change graph of the biomimetic nanoparticles selectively targeting PSMA in Example 1 under laser irradiation with repeated switching of the laser.

[0023] Figure 10Figure 1 is a graph of the results of MTT assay of 22RV1 cells with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0024] Figure 11 Figure 2 is a graph of the results of MTT assay of LnCap cells with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0025] Figure 12 Figure 3 is a graph of the results of MTT assay of Hacat cells with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0026] Figure 13 Figure 4 is a flow graph of cell binding with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0027] Figure 14 Figure 5 is a flow graph of competitive binding of the biomimetic nanoparticles selectively targeting PSMA in Example 1 with antibodies and cells.

[0028] Figure 15 Figure 6 is the results of tumor cell treatment with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0029] Figure 16 Figure 7 is the results of laser confocal of tumor cell treatment with the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0030] Figure 17 Figure 8 is a graph of the metabolism of the biomimetic nanoparticles selectively targeting PSMA in Example 1 in mice.

[0031] Figure 18 Figure 9 is a graph of the targeting of tumors of the biomimetic nanoparticles selectively targeting PSMA in Example 1 in mice. DETAILED DESCRIPTION

[0032] Example 1:

[0033] Near-infrared fluorescent water-soluble carbon dots: 1 mmol of NB, 1 mmol of EDTA-Na, 10 mL of water were added to a polytetrafluoroethylene-lined stainless steel reactor and reacted at 180 °C for 5 h under autogenous pressure, and the supernatant was filtered with a 0.22 μm filter membrane, and the filtrate was purified by dialysis bag (MWCO = 100 Da) for 12 h. Finally, the black powdery CDs were collected by freeze-drying. The apparent morphology of the CDs is shown in the scanning electron microscope image of Figure 1 , the transmission electron microscope image of Figure 2 . The carbon dots have an excitation wavelength of 630 nm and an emission wavelength of 665 nm in the near-infrared I region. The fluorescence spectrum of the carbon dots is shown in Figure 3 .

[0034] N-acrylamidoglutaric acid (AAGA) synthesis: Amino glutaric acid (0.075 mol) and sodium hydroxide (0.225 mol) were dissolved in 75 mL of deionized water, which was placed in an ice bath under vigorous stirring. Subsequently, 6.8 mL of acryloyl chloride and 15 mL of tetrahydrofuran were added dropwise. The reaction was further stirred at room temperature for 3.5 h. During the reaction, the pH was maintained at 7-8. Then, the pH of the mixture was adjusted to 11 with 2 M NaOH, and then washed with ethyl acetate to remove the organic phase. The pH of the aqueous phase was acidified to 2 with 6 M HC1, and then the aqueous phase was extracted again with ethyl acetate to obtain the extract. The extract was dried with magnesium sulfate, filtered, concentrated, and precipitated in n-hexane. Repeated precipitation was performed for further purification. The collected precipitate was dried in vacuum.

[0035] N-acrylamidoglutaric acid (AAGA) synthesis: Amino glutaric acid (0.075 mol) and sodium hydroxide (0.225 mol) were dissolved in 75 mL of deionized water, which was placed in an ice bath under vigorous stirring. Subsequently, 6.8 mL of acryloyl chloride and 15 mL of tetrahydrofuran were added dropwise. The reaction was further stirred at room temperature for 3.5 h. During the reaction, the pH was maintained at 7-8. Then, the pH of the mixture was adjusted to 11 with 2 M NaOH, and then washed with ethyl acetate to remove the organic phase. The pH of the aqueous phase was acidified to 2 with 6 M HC1, and then the aqueous phase was extracted again with ethyl acetate to obtain the extract. The extract was dried with magnesium sulfate, filtered, concentrated, and precipitated in n-hexane. Repeated precipitation was performed for further purification. The collected precipitate was dried in vacuum.

[0036] Nanoparticle preparation: The molar ratio of monomers N-isopropyl acrylamide, N-tert-butyl acrylamide, N-acrylamidoglutaric acid, N-acrylamidobutandioic acid and N,N'- methylenebisacrylamide was 43:35:10:10:2, and a mixed monomer aqueous solution was prepared with a total monomer concentration of 65 mM, and the solution system was 50 mL. 5 mg of near-infrared fluorescent water-soluble carbon dots were added. The mixed solution was ultrasonicated for 30 minutes, the round-bottom flask was sealed using a bottle plug and sealing film and purged with nitrogen for 30 min, 30 mg of initiator ammonium persulfate was added, assisted magnetic stirring, and the reaction was carried out at 65°C for 3 h. Finally, the biomimetic nanoparticles selectively targeting PSMA were obtained by water dialysis. The apparent morphology thereof is shown in Figure 4 Scanning electron microscope images and Figure 5 Transmission electron microscope images.

[0037] The nanoparticle and protein binding results are shown in Figure 6 As shown in the binding isotherm, the prepared polymer has high affinity and selectivity for the epitope polypeptide and biomarker, and the Kd of the inhibitor for the PSMA polypeptide is calculated to be 0.1 nM. DValues up to 4.67 x 10 -9 M, K D Values up to 4.43 x 10 -9 M, K D Values up to 2.31 x 10 -5 M, K -5 M, K D Values differ by 4 orders of magnitude Nanoparticles have high affinity and selectivity.

[0038] Figures 7-9 The photothermal performance and stability of the nanoparticles are as follows: the photothermal conversion efficiency is 31.2%.

[0039] Fluorescence imaging and cancer treatment of biomimetic nanoparticles selectively targeting PSMA:

[0040] 1. MTT method for determining in vitro cytotoxicity of nanoparticle-coated cells

[0041] MTT method was used to study the in vitro cytotoxicity of nanoparticles on 22RV1, LnCap, and Hacat cells. The specific process is as follows: cells (5000 cells per well) were cultured in a 96-well plate at 37°C and 5% CO2. After the cells adhered for 24 h, the cells were treated with nanoparticles at concentrations of 5, 25, 50, 75, 100, and 125 μg / mL. After 24 h of incubation, 20 μL of MTT solution (5.0 mg / mL) was added, and the cells were incubated for another 4 h. Then, 100 μL of DMSO was added to each well. The absorbance at 570 nm was measured, and the results are shown in Figures 10-12 At a high concentration of 125 μg / mL of nanoparticles, the cells still had 84.1% cell viability, indicating that the nanoparticles had high biological safety.

[0042] 2. Flow cytometry characterization of nanoparticle-cell binding

[0043] The cells were collected and diluted to 30,000 / mL. 2 mL of the diluted cells were centrifuged at 1000 rpm for 3 min to collect the cells. Then, 2 mL of nanoparticles at a concentration of 90 μg / mL were added for incubation for 3 h. After that, flow cytometry was used for detection, and the results are shown in Figure 13 As can be seen, the nanoparticles have much higher affinity for positive cells 22RV1 and Lncap than for negative Hacat cells.

[0044] 3. Nanoparticle blocking of tumor cell surface PSMA molecules and antibody binding function

[0045] Tumor cells were taken from the logarithmic growth phase in good condition at a concentration of 1 x 10 6After centrifugation, each tube of tumor cells was blocked with nanoparticles for 1 h in a centrifuge tube, and tumor cells without preparation treatment were used as experimental controls. After adding PSMA primary antibody solution diluted with 3% BSA (dilution ratio 1:500) and incubating at 37°C for 1 h, the cells were washed with PBS twice; then, fluorescent secondary antibody PE was added (dilution ratio 1:500), and the cells were incubated at 37°C in the dark for 1 h, and then washed with PBS twice. Flow cytometry was used to analyze the PSMA binding amount of the sample cells treated with different preparations, so as to further explore the function of the nanoparticles in blocking the PSMA molecules on the surface of the tumor cells. The nanoparticles can recognize and anchor to the PSMA molecules on the cell surface, Figure 14 The antibody-positive cells after nanoparticle blocking were 1.2%, which was lower than the positive rate of 84.6% of the positive antibody binding. Flow cytometry was used to verify that the artificial antibody significantly blocked the PSMA molecules on the surface of the tumor cells.

[0046] 4. MTT method for determining the treatment effect of nanoparticles on tumor cells

[0047] Cancer cells in the exponential growth phase were collected and inoculated into a 6-well plate at a concentration of 1×10 6 cells / well. After the cells adhered, 125 μg / mL of nanoparticles were added, and the cells were co-cultured for 24 h. Then, 20 μL of MTT solution (5.0 mg / mL) was added, and the cells were incubated for another 4 h. Then, 100 μL of DMSO was added to each well. The absorbance at 570 nm was measured, and the results are shown in Figure 15 The high survival rate of the control cells indicates that PBS, SA, and light alone do not cause damage to the cells. The cells in the nanoparticle and light groups showed varying degrees of cell death, and the cells in the nanoparticle and laser irradiation group were almost completely dead, indicating that the nanoparticles have good therapeutic effects.

[0048] 5. Study of cell killing power

[0049] Confocal method was used to study the toxic effect of artificial antibodies on tumor cells: 22RV1 cells (50000 cells / well) were inoculated in a confocal for 24 h, and the culture solution was discarded. The cells were further cultured with an artificial antibody solution, and after 24 h, the culture solution was discarded. The drug was exposed or not exposed to 808 nm laser (2.0 W / cm2, 10 min), and the culture solution was discarded. The subsequent operation steps were the same as above. Calcein-AM / PI staining: 22RV1 cells (1×10 6 cells / dish) were inoculated in a 6-well plate and cultured for 24 h. The culture solution was discarded, and then Calcein-AM and PI staining solution was added. The cells were stained in the dark for 30 min, and then washed with sterile PBS and imaged. Figure 16 It can be seen that the nanoparticles and light have good diagnosis and treatment effects.

[0050] 6. In vivo metabolism of nanoparticles

[0051] Fifteen mice were randomly divided into three groups (n=5 each) for experimental comparison: Group 1 received tail vein injection of PBS, Group 2 received carbon dots via tail vein and Group 3 received nanoparticles via tail vein. In vivo fluorescence imaging was performed at multiple time points (0h, 4h, 8h, 13h, 24h) post-injection to observe the distribution kinetics and tumor targeting selectivity of the nanoparticles. Figure 17 It can be seen that 2-4 h after injection of nanoparticles, enrichment can be seen in the liver, kidneys and a small amount of bladder. 8 to 12 hour images can see obvious kidney metabolism, and excreted out of the body by urine through the bladder.

[0052] 7. In vivo tumor targeting of nanoparticles

[0053] A 22RV1 xenograft mouse model was established by subcutaneous injection of 1 x 10 7 cells in the right shoulder of nude mice. When the tumor volume reached about 100mm 3

[0054] Fifteen mice were randomly divided into three groups (n=5 each) for experimental comparison: Group 1 received tail vein injection of PBS, Group 2 received carbon dots via tail vein and Group 3 received nanoparticles via tail vein. In vivo fluorescence imaging was performed at multiple time points (0h, 4h, 8h, 13h, 24h) post-injection to observe the distribution kinetics and tumor targeting selectivity of the nanoparticles. Figure 18 It can be seen that 2-4 h after injection of nanoparticles, enrichment can be seen in the liver, kidneys and a small amount of bladder. 8 to 12 hour images can see obvious kidney metabolism, and excreted out of the body by urine through the bladder.

Claims

1. A method for preparing biomimetic nanoparticles that selectively target PSMA, characterized in that: The preparation method comprises the following steps: preparing a mixed aqueous solution of N-isopropylacrylamide, N-tert-butylacrylamide, N-acrylamidoglutaric acid, N-acrylamidobutanedioic acid, and N,N'-methylenebisacrylamide in a molar ratio of 40-50:30-40:5-15:5-15:2, adding a loading substance, ultrasonically mixing, purging with nitrogen to expel air, adding an initiator, reacting with sealing and stirring at 50-80°C for 2-6 hours, and finally dialyzing with water to obtain biomimetic nanoparticles that selectively target PSMA. The initiator is ammonium persulfate, azobisisobutyronitrile or tetramethylethylenediamine; The loading substance is one or more of near-infrared fluorescent water-soluble carbon dots, fluorescein isothiocyanate, rhodamine B, paclitaxel, adriamycin, doxorubicin hydrochloride, and ferroferric oxide magnetic particles.

2. The preparation method according to claim 1, characterized in that described N The synthesis method of -acrylaminoglutaric acid is as follows: adjusting the pH value of the aminoglutaric acid aqueous solution to 12.5-13.5 with sodium hydroxide or potassium hydroxide, then adding it dropwise to the acryloyl chloride solution under vigorous stirring in an ice bath, reacting at room temperature for 2-12 hours, adjusting the pH value to 10-12 with sodium hydroxide or potassium hydroxide, washing with ethyl acetate to remove the organic phase; then adjusting the pH value to 0-2 with hydrochloric acid, extracting the aqueous phase with ethyl acetate to obtain an extract, drying the extract with magnesium sulfate, filtering, and precipitating in n-hexane to obtain N -Acrylamidoglutaric acid.

3. The preparation method according to claim 1, characterized in that The synthesis method of N-acrylamidobutanedioic acid comprises the following steps: adjusting the pH value of an aqueous solution of aminobutanedioic acid to 12.5-13.5 with sodium hydroxide or potassium hydroxide, then adding the solution dropwise to an acryloyl chloride solution under vigorous stirring and in an ice bath, reacting the solution at room temperature for 2-12 hours, adjusting the pH value to 10-12 with sodium hydroxide or potassium hydroxide, washing with ethyl acetate to remove the organic phase, then adjusting the pH value to 0-2 with hydrochloric acid, extracting the aqueous phase with ethyl acetate to obtain an extract, drying the extract with magnesium sulfate, filtering the extract, and precipitating the extract in n-hexane to obtain N-acrylamidobutanedioic acid.

4. The preparation method according to claim 2 or 3, characterized in that The solvent in the acryloyl chloride solution is one or more of ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide.

5. The preparation method according to claim 1, characterized in that The preparation method of the near-infrared fluorescent water-soluble carbon dots is as follows: Nile blue, disodium ethylenediaminetetraacetic acid and water are mixed and added to a hydrothermal reactor, reacted at 160-200°C for 5-12 hours, the supernatant is filtered, the filtrate is dialyzed for purification and then freeze-dried to obtain black powdered near-infrared fluorescent water-soluble carbon dots.

6. The preparation method according to claim 5, characterized in that The molar ratio of Nile blue to disodium edetate is 0.3-3.

7. Use of the biomimetic nanoparticles selectively targeting PSMA prepared according to the method of any one of claims 1 to 3, 5 and 6 in the preparation of prostate fluorescence imaging agents, prostate cancer photothermal therapeutic agents or photothermal antibacterial agents.

Images