Bionic nano-particles for selectively targeting PSMA as well as preparation method and application of bionic nano-particles

Abstract

The invention discloses a bionic nano-particle for selectively targeting PSMA as well as a preparation method and application of the bionic nano-particle. According to the invention, new functional monomers N-acryloylamino glutaric acid and N-acryloylamino succinic acid are designed and synthesized by simulating a glutamic acid substrate of carboxypeptidase, and a series of PSMA-targeting bionic nanoparticles with high affinity and selectivity to PSMA are synthesized by utilizing the new monomers. The nanoparticles have the advantages of low cost, higher stability, longer service life and stronger severe environment resistance, and overcome the defects of high cost, low preparation efficiency, long screening period, easiness in denaturation, difficulty in storage and the like of protein antibodies. The water-soluble carbon dots with near-infrared fluorescence and photo-thermal characteristics are synthesized, are combined with PSMA-targeted bionic nanoparticles and can replace protein antibodies, nuclide imaging molecules and the like to be used for targeted recognition of tumor cells and effective fluorescence imaging for early diagnosis of cancers, and the tumor cells are further killed through photo-thermal therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to a bionic nanoparticle selectively targeting PSMA and a preparation method and application thereof. Background Art

[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 cancers, while its physiological expression level is low in normal tissues (prostate, small intestine, salivary glands and lacrimal glands, and kidneys). The biological characteristics of PSMA's specific expression on the surface of prostate cancer cells make it of great 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 have shown certain clinical application prospects in molecular imaging diagnosis and targeted therapy of prostate cancer. However, PSMA monoclonal antibodies belong to large molecular protein substances, have poor permeability to prostate cancer tumor parenchyma, long circulation half-life, large side effects of in vivo application, and are prone to cause immune response. In addition, the production cost of monoclonal antibodies is high, and the biological activity is easily changed during storage, which limits their application in the diagnosis and treatment of prostate cancer. In view of this phenomenon, it is urgent to find a biological molecule that can combine diagnosis and therapeutic drugs. Summary of the invention

[0003] The purpose of the present invention is to overcome the numerous defects of the detection and treatment technology using substances such as radionuclides and labeled PSMA monoclonal antibodies, and to provide a bionic nanoparticle selectively targeting PSMA and a preparation method and application thereof.

[0004] A method for preparing near-infrared fluorescent water-soluble carbon dots is as follows: Nile blue, disodium ethylenediaminetetraacetic acid and water are mixed and added into a hydrothermal reactor, reacted at 160-200° C. for 5-12 h, the supernatant is filtered, the filtrate is dialyzed and purified, and then freeze-dried to obtain black powdery near-infrared fluorescent water-soluble carbon dots.

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

[0006] The preparation method of the bionic nanoparticles selectively targeting PSMA comprises 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 load, ultrasonically mixing, adding an initiator after nitrogen is passed to exhaust the air, reacting with sealing stirring at 50-80°C for 2-6 hours, and finally dialyzing with water to obtain bionic nanoparticles selectively targeting PSMA.

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

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

[0009] The synthesis method of N-acrylamidobutanedioic acid (AAAA) is as follows: adjusting the pH value of the aqueous solution of aminobutanedioic acid to 12.5-13.5 with sodium hydroxide or potassium hydroxide, then dropping it into an acryloyl chloride solution under vigorous stirring and ice bath, stirring 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-acrylamidobutanedioic 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 loading material 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.

[0012] The prepared bionic nanoparticles selectively targeting PSMA are used in the preparation of prostate fluorescence imaging agents, prostate cancer photothermal therapeutic agents or photothermal antibacterial agents.

[0013] Based on the carboxypeptidase-active simulating substrate of PSMA, the present invention designed and synthesized new functional monomer molecules N-acrylamidoglutaric acid and N-acrylamidobutanedioic acid, and synthesized PSMA-targeted biomimetic nanoparticles with high affinity and selectivity for a series of PSMA using the new monomers, with an affinity constant of 10 -9M. The bionic nanoparticles have low cost, high stability, long service life and strong resistance to harsh environments, overcoming the shortcomings of protein antibodies, such as high cost, low preparation efficiency, long screening cycle, easy variability and difficult preservation. In classical visible light fluorescence imaging, since both the excitation light and the emission light are in the wavelength range of 400-650 nm, the light absorption and scattering effects of biological tissues in this wavelength and the interference of autofluorescence seriously affect the in vivo imaging effect. The present invention synthesizes water-soluble carbon dots with near-infrared fluorescence and photothermal properties, which are combined with bionic nanoparticles targeting PSMA, and can replace protein antibodies, radionuclide imaging molecules, etc. for targeted identification of tumor cells and effective fluorescence imaging for early diagnosis of cancer, and further kill tumor cells through photothermal therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 3 is a scanning electron microscope image of the near-infrared fluorescent water-soluble carbon dots in Example 1.

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

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

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

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

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

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

[0021] Figure 8 3 is a temperature variation diagram of the bionic nanoparticles selectively targeting PSMA in Example 1 under irradiation with different laser powers.

[0022] Fig. 9 This is a temperature change diagram of the bionic nanoparticles selectively targeting PSMA in Example 1 when the laser is repeatedly switched on and off under laser irradiation.

[0023] Fig.10This is a graph showing the results of an MTT experiment on 22RV1 cells using biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0024] Fig.11 This is a graph showing the results of an MTT experiment on LnCap cells using biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0025] Fig.12 This is a graph showing the results of the MTT experiment on Hacat cells using biomimetic nanoparticles that selectively target PSMA in Example 1.

[0026] Fig.13 This is a flow cytometric graph of the cell binding of the biomimetic nanoparticles selectively targeting PSMA in Example 1.

[0027] Fig.14 This is a flow cytometric graph of the competitive binding between the biomimetic nanoparticles selectively targeting PSMA and antibodies and cells in Example 1.

[0028] Fig.15 This is the result of the tumor cell treatment using the bionic nanoparticles selectively targeting PSMA in Example 1.

[0029] Fig.16 This is the laser confocal result of the bionic nanoparticles selectively targeting PSMA in Example 1 for treating tumor cells.

[0030] Fig.17 This is the metabolic diagram of the biomimetic nanoparticles selectively targeting PSMA in Example 1 in mice.

[0031] Fig.18 This is a diagram of the bionic nanoparticles selectively targeting PSMA in Example 1 targeting tumors in mice. DETAILED DESCRIPTION

[0032] Embodiment 1: Near-infrared fluorescent water-soluble carbon dots: 1 mmol NB, 1 mmol EDTA-Na, and 10 mL water were added to a polytetrafluoroethylene-lined stainless steel reactor and reacted at 180 °C for 5 h under autogenous pressure. The supernatant was filtered with a 0.22 µm filter membrane, and the filtrate was dialyzed and purified for 12 h using a dialysis bag (MWCO = 100 Da). Finally, black powdered CDs were collected by freeze drying. Its appearance is shown in Figure 1 Scanning electron micrograph of Figure 2 Transmission electron microscope image of carbon dots. The excitation wavelength of carbon dots in the near-infrared I region is 630 nm, and the emission wavelength is 665 nm. Its fluorescence spectrum is shown in Figure 3 .

[0033] Synthesis of N-acryloylaminoglutaric acid (AAGA): Aminoglutaric acid (0.075 mol) and sodium hydroxide (0.225 mol) were dissolved in 75 mL of deionized water and placed in an ice bath under vigorous stirring. Subsequently, 6.8 mL of acryloyl chloride and 15 mL of tetrahydrofuran were added dropwise to the mixed solution. The reactants were further stirred at room temperature for 3.5 h. During the reaction, the pH value 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 value of the aqueous phase was acidified to 2 with 6 M HCl, and then the aqueous phase was extracted again with ethyl acetate to obtain an extract. The extract was dried over magnesium sulfate, filtered, concentrated, and precipitated in n-hexane. Repeated precipitation was performed for further purification. The collected precipitate was dried in a vacuum.

[0034] Synthesis of N-acryloylaminosuccinic acid (AAAA): Aminosuccinic acid (0.075 mol) and sodium hydroxide (0.225 mol) were dissolved in 75 mL of deionized water and placed in an ice bath under vigorous stirring. Subsequently, 6.8 mL of acryloyl chloride and 15 mL of tetrahydrofuran were added dropwise. The reactants were further stirred for 3.5 h at room temperature. During the reaction, the pH value 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 value of the aqueous phase was acidified to 2 with 6 M HCl, and then the aqueous phase was extracted again with ethyl acetate. The extract was dried over magnesium sulfate, filtered, concentrated, and precipitated in n-hexane. Repeated precipitation was performed for further purification. The collected precipitate was dried in a vacuum.

[0035] Preparation of nanoparticles: The molar ratio of monomers N-isopropylacrylamide, N-tert-butylacrylamide, N-acrylamidoglutaric acid, N-acrylamidobutanedioic acid and N,N'-methylenebisacrylamide is 43:35:10:10:2. A mixed monomer aqueous solution is prepared with a total monomer concentration of 65 mM and a solution system of 50 mL. 5 mg of near-infrared fluorescent water-soluble carbon dots are added. The mixed solution is sonicated for 30 minutes, the round-bottom flask is sealed with a stopper and a sealing film, and nitrogen is introduced for 30 min. 30 mg of initiator ammonium persulfate is added, and magnetic stirring is assisted. The reaction is carried out at 65°C for 3 h, and finally dialyzed with water to obtain bionic nanoparticles that selectively target PSMA. Its apparent morphology is shown in Figure 4 Scanning electron microscopy images and Figure 5 Transmission electron microscopy image.

[0036] The results of the determination of the binding between nanoparticles and proteins are as follows Figure 6 As shown in the isotherm curve, the prepared polymer has high affinity and selectivity for epitope peptides and biomarkers. The K of the inhibitor to PSMA peptide was calculated. DThe value can reach 4.67×10 -9 M, K for PSMA protein D The value can reach 4.43×10 -9 M, K for human serum albumin D Value 2.31×10 -5 M, for human myoglobin 2.09×10 -5 M, K compared to the magazine protein D The values ​​differ by 4 orders of magnitude, indicating that the nanoparticles have high affinity and selectivity.

[0037] Figure 7-9 It is the photothermal performance and stability of the nanoparticles, and the photothermal conversion efficiency is 31.2%. Fluorescence imaging and cancer therapy of biomimetic nanoparticles selectively targeting PSMA: 1. MTT assay to determine the in vitro cytotoxicity of nanoparticles

[0038] The MTT method was used to study the in vitro cytotoxicity of the nanoparticles on 22RV1, LnCap, and Hacat cells. The specific process was as follows: cells (5000 cells per well) were cultured in 96-well plates at 37°C and 5% CO 2 After the cells were attached for 24 h, the cells were treated with nanoparticles at concentrations of 5, 25, 50, 75, 100, and 125 μg / mL, respectively. After incubation for 24 h, 20 μL of MTT solution (5.0 mg / mL) was added, and incubated for another 4 h. 100 μL of DMSO was added to each well. The absorbance at 570 nm was measured. The results are shown in Figure 2. Figure 10-12 As shown, at a high concentration of 125 μg / mL of nanoparticles, the cells still had 84.1% cell viability, indicating that the nanoparticles have high biosafety. 2. Characterization of Nanoparticle-Cell Binding by Flow Cytometry

[0039] Collect the cells, dilute the cells to 30,000 / mL, take 2 mL of the diluted cells and centrifuge at 1000 rpm for 3 min to collect the cells, add 2 mL of 90 μg / mL nanoparticles and incubate for 3 h, then detect using flow cytometry. The results are as follows Fig.13 As shown, it can be seen that the nanoparticles have a much higher affinity for positive cells 22RV1 and Lncap than for negative Hacat cells. 3. Nanoparticles block the binding function of PSMA molecules on the surface of tumor cells and antibodies

[0040] Tumor cells in good logarithmic growth phase were selected and counted as 1×10 6After centrifugation, each tube was divided into groups and blocked with nanoparticles in a centrifuge tube for 1 hour. Tumor cells not treated with the preparation were used as the experimental control group. PSMA primary antibody solution diluted with 3% BSA (dilution ratio of 1:500) was added, incubated at 37°C for 1 hour, and then washed twice with PBS; fluorescent secondary antibody PE (dilution ratio of 1:500) was added, incubated at 37°C in the dark for 1 hour, and then washed twice with PBS. Cytometry was used to statistically analyze the PSMA binding amount of sample cells treated with different preparations, so as to further explore the function of nanoparticles in blocking PSMA molecules on the surface of tumor cells. Nanoparticles can recognize and anchor with PSMA molecules on the cell surface. Fig.14 The antibody-positive cells after nanoparticle blocking were 1.2%, which was lower than the positive rate of positive antibody binding of 84.6%. Flow cytometry was used to verify that the artificial antibodies significantly blocked the PSMA molecules on the surface of tumor cells. 4. MTT method to determine the therapeutic effect of nanoparticles on tumor cells

[0041] Cancer cells in the exponential growth phase were collected at a rate of 1×10 6 After the cells adhered to the wall, 125 μg / mL of nanoparticles were added. After 24 h of co-culture, 20 μL of MTT solution (5.0 mg / mL) was added. After another 4 h of incubation, 100 μL of DMSO was added to each well. The absorbance at 570 nm was measured. The results are as follows: Fig.15 As shown, the cells in the control group had a high survival rate, indicating that PBS, SA and light alone would not damage the cells. The cells in the nanoparticle and light-added groups showed different degrees of cell death, among which almost all the cells in the nanoparticle laser irradiation group died, and the nanoparticles had a good therapeutic effect. 5. Cell killing study

[0042] The confocal microscope was used to study the toxicity of artificial antibodies on tumor cells: 22RV1 cells (50,000 cells / well) were inoculated in the confocal microscope and cultured for 24 h, the culture medium was discarded, and the culture medium was further cultured with artificial antibody. After 24 h, the culture medium was discarded, and the drugs were exposed or not exposed to 808 nm laser (2.0 W / cm2, 10 min), and the culture medium was discarded. The subsequent operation steps were the same as above. Calcein-AM / PI staining: 22RV1 cells (1×10 6 The cells were inoculated into 6-well plates (100 cells / dish) and cultured for 24 h. The culture medium was discarded, and Calcein-AM and PI staining solutions were added. The cells were stained in the dark for 30 min, and then washed with sterile PBS and imaged. Fig.16 It can be seen that nanoparticles plus light have good diagnostic and therapeutic effects. 6. In vivo metabolism of nanoparticles

[0043] Fifteen mice were randomly divided into three groups (n=5 per group) for experimental comparison: Group 1 received PBS injection via tail vein, 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 after injection (0 h, 4 h, 8 h, 13 h, 24 h) to observe the distribution dynamics and tumor targeting selectivity of the nanoparticles. Fig.17 It can be seen that the nanoparticles are enriched in the liver, kidneys and a small amount of bladder 2-4 hours after injection. The images from 8 to 12 hours show obvious renal metabolism and excretion from the body through the bladder and urine. 7. In vivo tumor targeting of nanoparticles

[0044] The nude mice were injected subcutaneously with 1 × 10 7 The 22RV1 xenograft mouse model was established with 100 cells. 3 At 14:50 pm, mice were randomly divided into three groups (n=5 per group) for experimental comparison: Group 1 received PBS injection via tail vein, 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 (0 h, 4 h, 8 h, 13 h, 24 h) after injection to observe the distribution dynamics and tumor targeting selectivity of nanoparticles. Fig.18 It can be seen that the nanoparticles are enriched in the liver, kidneys and a small amount of bladder 2-4 hours after injection. The images from 8 to 12 hours show obvious renal metabolism and excretion from the body through the bladder and urine.

Claims

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

2. The preparation method according to claim 1, characterized in that: The molar ratio of Nile blue to disodium ethylenediaminetetraacetate is 0.3-3.

3. A method for preparing biomimetic nanoparticles selectively targeting PSMA, characterized in that: The specific operation of the preparation method is: 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 load, ultrasonically mixing, adding an initiator after nitrogen is passed to exhaust the air, reacting with sealing stirring at 50-80°C for 2-6 hours, and finally dialyzing with water to obtain bionic nanoparticles that selectively target PSMA.

4. The preparation method according to claim 3, characterized in that: The initiator is ammonium persulfate, azobisisobutyronitrile or tetramethylethylenediamine.

5. The preparation method according to claim 3, characterized in that: Said N The synthesis method of -acrylaminoglutaric acid is as follows: adjust the pH value of the aminoglutaric acid aqueous solution to 12.5-13.5 with sodium hydroxide or potassium hydroxide, then add it dropwise to the acryloyl chloride solution under vigorous stirring and ice bath, react at room temperature for 2-12 hours, adjust the pH value to 10-12 with sodium hydroxide or potassium hydroxide, wash and remove the organic phase with ethyl acetate; then adjust the pH value to 0-2 with hydrochloric acid, extract the aqueous phase with ethyl acetate to obtain an extract, dry the extract with magnesium sulfate, filter and precipitate in n-hexane to obtain N -Acryloylaminoglutaric acid.

6. The preparation method according to claim 3, characterized in that: The synthesis method of N-acryloylaminosuccinic acid comprises the following steps: adjusting the pH value of an aqueous solution of aminosuccinic acid to 12.5-13.5 with sodium hydroxide or potassium hydroxide, then dropping the solution into an acryloyl chloride solution under vigorous stirring and in an ice bath, stirring 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 an 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 the extract in n-hexane to obtain N-acryloylaminosuccinic acid.

7. The preparation method according to claim 5 or 6, characterized in that: The solvent in the acryloyl chloride solution is one or more of ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide.

8. The preparation method according to claim 3, characterized in that: The loading material 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.

9. Use of the bionic nanoparticles selectively targeting PSMA prepared according to the method of any one of claims 3 to 6 and 8 in the preparation of prostate fluorescent imaging agents, prostate cancer photothermal therapeutic agents or photothermal antibacterial agents.

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