Preparation method and application of polymer nanomedicine targeting somatostatin receptor 2

Abstract

The present invention relates to a preparation method and application of a polymer nano drug targeting somatostatin receptor 2, effectively solving the problem of preparing a polymer nano drug targeting somatostatin receptor 2, and its application in preparing a drug for treating neuroendocrine prostate cancer, wherein PLGA-PEG-Mal is dissolved in dichloromethane, double distilled water is added, and the suspension is initially emulsified, and then a sodium cholate aqueous solution is added for double emulsion, and the suspension after the two emulsifications is added dropwise to the sodium cholate aqueous solution, and the suspension is dispersed by magnetic stirring, and the dichloromethane in the solution is removed by rotary evaporation under reduced pressure, centrifuged, and the precipitate is washed twice with double distilled water, and then the precipitate is resuspended in double distilled water to obtain a PLGA nanoparticle suspension; octreotide is mixed with the PLGA nanoparticle suspension, double distilled water or PBS buffer is added, and the suspension is placed in a suspension instrument for reaction, dialyzed, and unconnected octreotide is removed to obtain a polymer nano drug targeting somatostatin receptor 2. The method of the present invention is easy to operate, has a scientific and reasonable design, and has good performance of the prepared product.

Description

Technical Field

[0001] The invention relates to medicine, in particular to a preparation method of a polymer nano drug targeting somatostatin receptor 2 and application thereof. Background Art

[0002] Neuroendocrine prostate cancer is the most malignant pathological subtype of prostate cancer, characterized by low expression of androgen receptors and high expression of neuroendocrine markers. Neuroendocrine prostate cancer is insensitive to androgen deprivation therapy, chemotherapy, and radiotherapy, and is currently one of the main causes of prostate cancer-specific death. To date, no effective treatment for neuroendocrine prostate cancer has been provided, and no anti-neuroendocrine prostate cancer drugs have been approved. Therefore, finding a safe and effective anti-tumor strategy is crucial to improving the prognosis of patients with neuroendocrine prostate cancer.

[0003] Poly(lactic-co-glycolic acid) (PLGA), as a biodegradable nanocarrier, has been approved by the FDA as an excipient for the production and processing of various drugs. As early as 1989, PLGA-based leuprolide sustained-release microspheres have been used in long-term endocrine therapy for prostate cancer patients and are still used today. In addition, the surface of PLGA carriers can also be modified with different functions to target the delivery of therapeutic drugs to specific cells or organs. Somatostatin receptor 2 (SSTR2) is highly expressed on the cell membrane of neuroendocrine tumors, and octreotide (Oct), as an artificially synthesized octapeptide derivative of natural somatostatin, can bind to SSTR2. Therefore, octreotide can be used as a targeting peptide for the construction of drug delivery carriers for neuroendocrine tumors.

[0004] In addition, serine / threonine PKCβ is believed to be involved in the progression of neuroendocrine prostate cancer. Enza, as an inhibitor of PKCβ, can inhibit the proliferation of neuroendocrine prostate cancer and induce its apoptosis by inhibiting the expression and activity of PKCβ. However, several clinical studies have found that blood exposure to enzastaurin increases the occurrence of toxic side effects such as thrombosis, thrombocytopenia, bleeding, and elevated alanine aminotransferase. Therefore, efficient delivery of enzastaurin to neuroendocrine prostate cancer cells, improving its bioavailability and reducing the occurrence of adverse complications is an effective strategy for the treatment of neuroendocrine prostate cancer. However, there has been no public report on the use of octreotide as a targeting ligand modified on the surface of PLGA carriers and loaded with PKCβ inhibitors. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for preparing a polymer nanodrug targeting somatostatin receptor 2 and its application, which can effectively solve the problem of preparing polymer nanodrugs targeting somatostatin receptor 2 and its application in the preparation of drugs for the treatment of neuroendocrine prostate cancer.

[0006] The technical solution provided by the present invention is a method for preparing a polymer nano drug targeting somatostatin receptor 2, wherein the PLGA carrying the PKCβ inhibitor is synthesized by the double emulsion method, the bioavailability of enzastaurin is greatly improved, and octreotide is modified on the surface of PLGA by chemical coupling reaction of maleimide bond and thiol. The modification of octreotide increases the enrichment of the nano drug in neuroendocrine tumor tissue, and can effectively inhibit the proliferation, invasion and migration ability of neuroendocrine prostate cancer cells, thereby achieving the purpose of anti-tumor, which is achieved by the following method:

[0007] 1. Preparation of PLGA nanoparticles:

[0008] 1) Weigh 10 mg of PLGA-PEG-Mal and dissolve it in 1 mL of dichloromethane;

[0009] The mass ratio of PLGA, PEG and Mal in the PLGA-PEG-Mal is 2000:17000:97;

[0010] 2) Add 200 μL of double distilled water and use an ultrasonic disruptor for primary emulsification. Set the power to 285 W, turn on for 1 second, turn off for 2 seconds, and continue for 3 minutes to form a suspension;

[0011] 3) Add 2 mL of 2% sodium cholate aqueous solution to the suspension after the initial emulsification, and use an ultrasonic disruptor for re-emulsification, with the power set to 332.5 W, on for 1 second, off for 2 seconds, and lasting for 5 minutes;

[0012] 4) The twice emulsified suspension was added dropwise into 10 mL of a 0.5% sodium cholate aqueous solution and dispersed using a magnetic stirrer for 15 min;

[0013] 5) Under reduced pressure, the dispersed solution was evaporated using a rotary evaporator until no bubbles were generated, thereby removing the dichloromethane in the solution;

[0014] 6) centrifuging the clear transparent solution obtained in step 5) at 10,000 g, room temperature, for 15 min;

[0015] 7) discard the supernatant, wash the precipitate twice with double distilled water, repeat step 6), resuspend the precipitate at the bottom of the tube in 1 mL of double distilled water to obtain a PLGA nanoparticle suspension;

[0016] 2. Preparation of octreotide-modified PLGA nanocarriers:

[0017] 1) Octreotide and PLGA nanoparticles were mixed at a molar ratio of 2:1;

[0018] 2) Take double distilled water or PBS buffer, pH 6.5-8.0;

[0019] 3) adding the mixture of octreotide and PLGA into double distilled water or PBS buffer at a volume ratio of 1:1, and reacting in a suspension instrument at 18-25° C., 300 rpm, for 3-12 hours;

[0020] 4) Select a dialysis bag with a molecular weight cutoff of 2000D-5000D and activate it by boiling in double distilled water for 3-5 minutes;

[0021] 5) adding the solution obtained in 3) into an activated dialysis bag, sealing it with sealing clips at the top and bottom, immersing it in a glass beaker filled with double distilled water, and dialyzing it with stirring overnight for 8-12 hours to remove the unattached octreotide;

[0022] 6) The solution obtained after dialysis is the octreotide-modified PLGA nanocarrier (Oct-PLGA), that is, the polymer nanomedicine targeting somatostatin receptor 2.

[0023] The polymer nano drug (Oct-PLGA) targeting somatostatin receptor 2 prepared by the method of the present invention has anti-cancer activity and can be used in the preparation of drugs for treating neuroendocrine prostate cancer.

[0024] The method of the invention is easy to operate, has a scientific and reasonable design, and the prepared product has good performance. It is effectively used for preparing drugs for treating neuroendocrine prostate cancer, opens up a new way for drugs for treating neuroendocrine prostate cancer, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscope image of the nanomedicine of the present invention.

[0026] Figure 2 The figure shows the hydrated particle size and surface potential of the nano drug of the present invention.

[0027] Figure 3 These are the NMR images of the nano drug octreotide before and after modification.

[0028] Figure 4 Schematic diagram of the targeting of nanoparticles of the present invention.

[0029] Figure 5 This is a diagram showing that the nanomedicine of the present invention inhibits the proliferation of neuroendocrine prostate cancer cells.

[0030] Figure 6 This is a graph showing the ability of the nanomedicine of the present invention to inhibit the invasion of neuroendocrine prostate cancer cells.

[0031] Figure 7 This is a graph showing the ability of the nanomedicine of the present invention to inhibit the migration of neuroendocrine prostate cancer cells. DETAILED DESCRIPTION

[0032] The specific implementation modes of the present invention are described in detail below in conjunction with the embodiments.

[0033] Example 1

[0034] The present invention provides a method for preparing a polymer nano drug targeting somatostatin receptor 2, comprising the following steps:

[0035] (1) Preparation of PLGA-Enza nanodrugs:

[0036] Weigh 10 mg of PLGA-PEG-Mal and 1 mg of enzastaurin and dissolve them in 1 mL of dichloromethane; add 200 μL of double distilled water, use an ultrasonic crusher for primary ultrasonic emulsification, set the power to 285 W, turn on for 1 s, turn off for 2 s, and last for 5 minutes; add 2 mL of 2% sodium cholate aqueous solution, use an ultrasonic crusher for secondary ultrasonic emulsification, set the power to 332.5 W, turn on for 1 s, turn off for 2 s, and last for 5 minutes; add the ultrasonicated liquid drop by drop into the 10 mL mass concentration The suspension was evaporated by a rotary evaporator under a negative pressure of 2Kpa-10Kpa until no bubbles appeared, and the dichloromethane in the solution was removed; the obtained clear and transparent solution was centrifuged in a centrifuge at 10000g for 15min at room temperature; after the centrifugation, the supernatant was discarded, the precipitate was washed twice with double distilled water, and centrifuged again, and the obtained nanoparticles (PLGA-Enza) were resuspended in 1mL water for later use;

[0037] (2) Preparation of Oct-PLGA-Enza nanodrugs:

[0038] PLGA-Enza and octreotide are mixed in a molar ratio of 1:2, double distilled water or PBS buffer is added, the pH value is adjusted to 6.5-8.0, and the mixture is placed on a mixer at 300 rpm and mixed at room temperature for 8 hours to obtain a mixed solution; a dialysis bag with a molecular weight cutoff of 3500D is selected, and it is boiled and activated in double distilled water for 4 minutes. The mixed solution is added to the activated dialysis bag, which is sealed with sealing clips at the top and bottom, and immersed in a glass beaker filled with double distilled water, and dialyzed for 10 hours while stirring overnight to remove unconnected octreotide. The solution obtained after dialysis is octreotide-modified nanodrug particle polymer (Oct-PLGA-Enza), that is, a polymer nanodrug targeting somatostatin receptor 2.

[0039] The present invention uses octreotide as a targeting ligand to modify the surface of the PLGA carrier and carry the PKCβ inhibitor. Its advantages are: (1) It solves the problem of low bioavailability of enzastaurin; (2) It reduces the nonspecific uptake of enzastaurin by other cells in the body and reduces the toxic and side effects of the drug; (3) It is easy to synthesize and biodegradable; (4) It can be loaded with anti-tumor drugs of different mechanisms and flexibly combined according to clinical needs. The experiment has achieved very good beneficial technical effects. The relevant experimental data are as follows:

[0040] 1. Determination of encapsulation efficiency and drug loading of Oct-PLGA-Enza nanodrugs: First, the maximum absorption wavelength of Enzatoline was determined to be 255nm by an ELISA instrument. Then, the Enzatoline solutions of different concentration gradients were measured by liquid-high performance chromatography and a standard curve was drawn. Finally, the area under the maximum absorption peak of Enzatoline in the loaded nanodrug was measured, and the content of Enzatoline was determined according to the drawn standard curve. The encapsulation efficiency was calculated as follows: Encapsulation efficiency (%) = drug mass encapsulated by nanoparticles / total mass of the input drug * 100%, and the drug loading was calculated as follows: Drug loading (%) = drug mass encapsulated by nanoparticles / (total mass of the input drug + total mass of the input PLGA) × 100%. The results are shown in Table 1.

[0041] Table 1 Encapsulation efficiency (EE) and drug loading rate (DLR) of nanocarriers, the mass of PLGA is fixed at 10 mg

[0042]

[0043] 2. Experiment on modification of octreotide on PLGA-Enza via thiol-maleimide coupling reaction:

[0044] 1 mg of PLGA and Oct-PLGA nanoparticles were prepared according to the above method, freeze-dried in a low-temperature freeze dryer, dissolved in 0.5 mL of deuterated DMSO, placed in a nuclear magnetic resonance instrument for detection, and the results were interpreted using MestReNova 9.0 software. Figure 3 As shown: The disappearance of maleimide protons (6.67 ppm) in the Oct-PLGA NMR results proves the successful coupling of octreotide to PLGA-PEG-Mal.

[0045] 3. Nanoparticle neuroendocrine prostate cancer cell targeting experiment:

[0046] The amino acids constituting octreotide were randomly arranged in scrambled order to synthesize mutant octreotide (mOct), which was used as a control peptide for subsequent experiments. Subsequently, Cy5.5 was selected as a fluorescent dye and three groups of nanoparticles were synthesized according to the above method: PLGA-Cy5.5, Oct-PLGA-Cy5.5 and mOct-PLGA-Cy5.5.

[0047] H660 or PC3 cells were seeded into 12-well plates at an appropriate density; cultured for 48-72 hours, the culture medium was discarded, PBS was slowly added to wash the cells, and new culture medium was replaced (considering the semi-adherent and semi-suspended characteristics of H660, the suspended cells were added to the well plate again after centrifugation); the three nanoparticles prepared above that were loaded with equal amounts of fluorescent dye Cy5.5 were added to the well plate and incubated on ice for 30 minutes; trypsin digestion was performed, and complete culture medium was used to terminate the digestion. The cells were centrifuged at a centrifugal force of 300g for 3-5 minutes, and the cells were collected and centrifuged in a 1.5mL EP tube. After the supernatant was discarded, 200μL PBS was added to resuspend the cells; flow cytometer detection was performed. Figure 4 Shown: Octreotide-modified polymer nanoparticles have good targeting properties to neuroendocrine prostate cancer cells.

[0048] IV. Experiment on the inhibitory effect of polymer nanomedicine on the proliferation of neuroendocrine prostate cancer cells detected by CCK-8 method

[0049] H660 or PC3 cells were seeded in a 96-well plate at an appropriate density, placed in a cell culture incubator, and cultured for 24-48 hours. Oct-PLGA-Enza nanodrugs were synthesized according to the above method, and three treatment groups were set up: a blank control group, an enzastaurin free drug group, and an Oct-PLGA-Enza nanodrug group. The final experimental concentration of enzastaurin was set to 20 μM. After giving corresponding treatments according to the groups, the cells were incubated for another 24 hours, the culture medium was carefully aspirated, and complete culture medium containing 10% CCK-8 was added, and the cells were placed in a 37°C cell culture incubator for incubation for 1 hour; the 96-well plate was taken out, and the OD value at 450 nm was detected by an enzyme reader. Figure 5 As shown: Oct-PLGA-Enza nanodrug can significantly inhibit the proliferation of neuroendocrine prostate cancer cells.

[0050] 5. Transwell assay to detect the inhibitory effect of polymer nanomedicine on the invasion ability of neuroendocrine prostate cancer cells

[0051] The Transwell chamber was prepared by mixing Matrigel matrix gel and 1640 culture medium in a volume ratio of 1:3, and gently shaken to make it evenly distributed, with a thickness of about 0.5-1mm, and placed in a 37°C incubator for 1h. H660 or PC3 cells with good growth status were selected and grouped: blank control group, enzastaurin free drug group and Oct-PLGA-Enza nanodrug group. Oct-PLGA-Enza nanodrugs were synthesized according to the above method, and the final experimental concentration of enzastaurin was set to 20μM. After the cells were treated accordingly according to the grouping, they were added to the Transwell chamber, and 600μl of complete culture medium containing 10% FBS was added to the small lower hole of the Transwell chamber (ensuring that the chamber is in full contact with the culture medium and there are no bubbles in the middle), and the cells were incubated at 37°C incubator (5% CO 2 ) overnight. After overnight culture, take out the Transwell chamber, remove the liquid inside, gently wipe the inner surface of the chamber with a cotton swab to remove residual cells and culture medium, place it in a new well, and wash it twice with PBS, each time for 5 minutes. Add an appropriate amount of 4% paraformaldehyde solution to the new well, immerse the chamber in it for fixation, and wash it twice with PBS solution after 20 minutes, each time for 5 minutes. Add an appropriate amount of 5% Ttrixon to the new well, immerse the chamber in it for 5 minutes, wash it twice with PBS, each time for 5 minutes, and dry the chamber membrane at room temperature. Prepare 0.1% crystal violet staining solution with 1x PBS, and measure 600μl into a new well. Under light-proof conditions, immerse the dried chamber in the staining solution and stain for 10 minutes. Wash it twice with PBS, each time for 5 minutes, and after drying at room temperature, take pictures and samples under a microscope. Take 3 fields of view in each well, and calculate the number of cells that have penetrated the mold. The above experiment was repeated 3 times, and statistical analysis was performed using SPSS2.0 software. Figure 6 As shown: Oct-PLGA-Enza nanodrug can significantly inhibit the invasive ability of neuroendocrine prostate cancer cells.

[0052] VI. Scratch test to detect the inhibitory effect of polymer nanomedicine on the migration ability of neuroendocrine prostate cancer cells

[0053] PC3 cells were seeded at an appropriate density in a 6-well plate, placed in a cell culture incubator, and cultured for 24 hours. After the cells adhered to the wall, a small or medium-sized sterile pipette tip was selected, the pipette tip was kept vertical, and a ruler was used to guide the pipette tip to make a single scratch in one direction perpendicular to the horizontal line on the back of the 6-well plate. Three scratches could be made in each well as a repeat. Three treatment groups were set up: a blank control group, an enzastaurin free drug group, and an Oct-PLGA-Enza nanodrug group. The Oct-PLGA-Enza nanodrug was synthesized according to the aforementioned method, and the final experimental concentration of enzastaurin was set to 20 μM. After the corresponding treatments were given according to the groups, they were cultured for 1-2 days, and samples were taken under an inverted microscope at 0 hours, 24 hours, and 48 hours. Image J software was used to compare and analyze the changes in the cell scratch spacing between the two groups at each time point to draw the final conclusion. Figure 7 As shown: Oct-PLGA-Enza nanodrug can significantly inhibit the migration ability of neuroendocrine prostate cancer cells.

[0054] Experiments show that the polymer nanomedicine (Oct-PLGA-Enza) targeting somatostatin receptor 2 prepared by the method of the present invention has anti-cancer activity, can be effectively used to treat neuroendocrine prostate cancer, and can be used in the preparation of drugs for treating neuroendocrine prostate cancer. It effectively solves the problem of low bioavailability of enzastaurin, reduces the nonspecific uptake of enzastaurin by other cells in the body, reduces drug toxicity and side effects, is simple to synthesize, is biodegradable, can be loaded with anti-tumor drugs of different mechanisms, and can be flexibly combined according to clinical needs, opening up a new way of treating neuroendocrine prostate cancer drugs, and has significant economic and social benefits.

Claims

1. A method for preparing a polymer nanodrug targeting somatostatin receptor 2, It is characterized in that The following steps are involved:

1. Preparation of PLGA nanoparticles: 1) Weigh 10 mg of PLGA-PEG-Mal and dissolve it in 1 mL of dichloromethane; The mass ratio of PLGA, PEG and Mal in the PLGA-PEG-Mal is 2000:17000:97; 2) Add 200 μL of double distilled water and use an ultrasonic disruptor for primary emulsification. Set the power to 285 W, turn on for 1 second, turn off for 2 seconds, and continue for 3 minutes to form a suspension. 3) Add 2 mL of 2% sodium cholate aqueous solution to the initially emulsified suspension, and use an ultrasonic disruptor for re-emulsification, with the power set to 332.5 W, on for 1 second, off for 2 seconds, and lasting for 5 minutes; 4) Add the twice emulsified suspension dropwise into 10 mL of 0.5% sodium cholate aqueous solution and disperse it with a magnetic stirrer for 15 minutes; 5) Under reduced pressure, use a rotary evaporator to evaporate the dispersed solution until no bubbles are generated, and remove the dichloromethane in the solution; 6) Centrifuge the clear and transparent solution obtained in step 5) at 10,000 g, room temperature, for 15 min; 7) Discard the supernatant, wash the precipitate twice with double distilled water, repeat step 6), and resuspend the precipitate at the bottom of the tube in 1 mL of double distilled water to obtain a PLGA nanoparticle suspension; 2. Preparation of octreotide-modified PLGA nanocarriers: 1) Octreotide and PLGA nanoparticles were mixed at a molar ratio of 2:1; 2) Take double distilled water or PBS buffer, pH 6.5-8.0; 3) Add the mixture of octreotide and PLGA into double distilled water or PBS buffer at a volume ratio of 1:1, and react in a suspension instrument at 18-25°C, 300 rpm, for 3-12 hours; 4) Select a dialysis bag with a molecular weight cutoff of 2000D-5000D and activate it by boiling in double distilled water for 3-5 minutes; 5) Add the solution obtained in 3) to the activated dialysis bag, seal it with sealing clips at the top and bottom, immerse it in a glass beaker filled with double distilled water, and dialyze it overnight with stirring for 8-12 hours to remove the unattached octreotide; 6) The solution obtained after dialysis is the octreotide-modified PLGA nanocarrier Oct-PLGA.

2. A method for preparing a polymer nanomedicine targeting somatostatin receptor 2, It is characterized in that The following steps are involved: (1) Preparation of PLGA-Enza nanodrugs: Weigh 10 mg of PLGA-PEG-Mal and 1 mg of enzastaurin and dissolve them in 1 mL of dichloromethane; add 200 μL of double distilled water, use an ultrasonic crusher for primary ultrasonic emulsification, set the power to 285 W, turn on for 1 s, turn off for 2 s, and last for 5 minutes; add 2 mL of 2% sodium cholate aqueous solution, use an ultrasonic crusher for secondary ultrasonic emulsification, set the power to 332.5 W, turn on for 1 s, turn off for 2 s, and last for 5 minutes; add the ultrasonicated liquid drop by drop into 10 mL of concentrated sodium cholate solution. The suspension was evaporated by a rotary evaporator under a negative pressure of 2Kpa-10Kpa until no bubbles appeared, and the dichloromethane in the solution was removed; the obtained clear and transparent solution was centrifuged in a centrifuge at 10000g for 15min at room temperature; after the centrifugation, the supernatant was discarded, the precipitate was washed twice with double distilled water, and centrifuged again, and the obtained PLGA-Enza nanodrug was resuspended in 1mL water for standby use; (2) Preparation of Oct-PLGA-Enza nanodrugs: PLGA-Enza and octreotide were mixed in a molar ratio of 1:2, and double distilled water or PBS buffer was added to adjust the pH value to 6.5-8.0, and the mixture was placed on a mixer at 300 rpm and mixed at room temperature for 8 hours to obtain a mixed solution; a dialysis bag with a molecular weight cutoff of 3500D was selected, and it was boiled and activated in double distilled water for 4 minutes, and the mixed solution was added to the activated dialysis bag, which was sealed with sealing clips at the top and bottom, and immersed in a glass beaker filled with double distilled water, and dialyzed for 10 hours under stirring overnight to remove the unconnected octreotide. The solution obtained after the dialysis was the octreotide-modified nano drug particle polymer Oct-PLGA-Enza.

3. Use of the polymer nanomedicine targeting somatostatin receptor 2 prepared by the method according to any one of claims 1 to 2 in the preparation of a drug for treating neuroendocrine prostate cancer.

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