Method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles

Abstract

This invention relates to the field of biomedical technology and discloses a method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles. The method includes vessel pretreatment, gold nanoparticle synthesis, ligand modification, CTB coupling, and drug loading. By using mercaptosuccinic acid for surface modification in an alkaline pH environment, a stable carboxylation modification layer is formed on the surface of the gold nanoparticles, which improves the colloidal stability of the nanoparticles, prevents them from agglomerating in the physiological environment, and provides active sites for subsequent coupling of functional molecules. By using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and n-hydroxysuccinamide to catalyze a covalent coupling reaction, the binding activity of CTB on the GM1 ganglioside receptor is fully preserved, enabling it to recognize neuronal surface receptors and achieve efficient transaxonal retrograde transport of nanomedicines.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles. Background Technology

[0002] Spinal cord injury is the most serious complication of spinal cord injury, often leading to severe functional impairment of the limbs below the injured segment. Spinal cord injury not only causes serious physical and psychological harm to the patient, but also imposes a huge economic burden on the whole society.

[0003] Currently, although existing technologies have attempted to use nanocarriers to deliver therapeutic drugs to intervene in the process of spinal cord injury, conventional nanomedicines have difficulty crossing the axonal barrier to achieve retrograde transport from the distal end of the injury to the neuronal cell body. They also suffer from problems such as insufficient colloidal stability leading to in vivo aggregation, low coupling efficiency with functional molecules resulting in loss of targeting activity, low drug loading rate, and residual impurities during purification affecting the uniformity of the formulation. Therefore, they cannot effectively meet the treatment needs of diseases such as spinal cord injury.

[0004] Therefore, a method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles is proposed to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles, which solves the problems mentioned in the background technology, such as insufficient colloidal stability leading to in vivo aggregation, low coupling efficiency with functional molecules resulting in loss of targeting activity, low drug loading rate, and residual impurities during purification affecting the uniformity of the formulation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles, comprising the following steps:

[0007] Step 1: Pretreatment of the vessels. Immerse the round-bottom flask, glass stopper and magnetic stir bar in aqua regia solution for 25-35 minutes. After taking them out, rinse them with ultrapure water 3-5 times in sequence, and place them in an electric heating drying oven at 70±5℃ for 2-3 hours to dry.

[0008] Step 2: Synthesis of gold nanoparticles. Add 20 mL of an aqueous solution containing gold chloro acid to a pretreated round-bottom flask. The concentration of HAuCl4 in the gold chloro acid aqueous solution is 2.4 × 10⁻⁶. -4 -2.6×10 -4 The concentration of sodium tricitrate was 2.4 × 10 mol / L. -4 -2.6×10 -4At a stirring rate of 800-1200 r / min, 0.55-0.65 mL of a pre-cooled NaBH4 solution with a concentration of 0.09-0.11 mol / L was added at once, and stirring was continued until the solution color changed from light yellow to wine red to obtain the initial solution of gold nanoparticles.

[0009] Step 3: Ligand modification. The pH of the initial gold nanoparticle solution was adjusted to 10.5-11.5 with 0.08-0.12 mol / L NaOH solution. Mercaptosuccinic acid was added, with a molar ratio of mercaptosuccinic acid to HAuCl4 of 1.8-2.2:1. The mixture was stirred at 400-600 r / min for 10-14 h at room temperature. The solution was then washed with deionized water and subjected to ultracentrifugation to obtain a concentrated gold nanoparticle solution with a concentration of 2.4-2.6 mg / mL.

[0010] Step 4: CTB coupling. Take 2.4-2.6 mg of cholera toxin B subunit, 4.8-5.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2.4-2.6 mg of n-hydroxysuccinimide and dissolve them in 0.09-0.11 mol / L MES buffer. Mix with gold nanoparticle concentrate and stir at 200-400 r / min for 45-75 min at 4±2℃. After the reaction is completed, wash with ultrapure water at 4±2℃ and centrifuge at 10000-12000 g / min to obtain AuNP-CTB complex.

[0011] Step 5: Drug loading. Dilute the AuNP-CTB complex with PBS to 4.8-5.2 mg (Au) / mL, add 1 mL of DMSO solution containing 4.8-5.2 mg rolipran and 5 mL of DMSO solution containing 1.6-1.8 mL 4-dimethylaminopyridine, and stir at 300-500 r / min for 38-42 h at 25±2℃ in the dark. The reaction product is washed with ultrapure water and collected to obtain the transaxonal retrograde nanomedicine based on gold nanoparticles.

[0012] Preferably, the aqua regia solution in step one is prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1, and the temperature of the aqua regia solution is controlled at 20-25℃.

[0013] Preferably, the preparation method of the aqueous solution of chloroform in step two is as follows: dissolve solid HAuCl4 in ultrapure water, ultrasonically disperse for 5-10 min, then add sodium tricitrate, and continue ultrasonic dispersion for 10-15 min, with an ultrasonic power of 200-300W.

[0014] Preferably, the method for preparing the NaBH4 pre-cooled solution in step two is as follows: dissolve NaBH4 solid in deionized water pre-cooled at 0-4℃, prepare and use immediately, and the time interval from preparation to addition of gold chloro acid aqueous solution shall not exceed 5 minutes.

[0015] Preferably, the parameters for ultracentrifugation in step three are: rotation speed 12000-15000 g / min, centrifugation time 15-20 min, centrifugation temperature 4-8℃, and repeated centrifugation 2-3 times.

[0016] Preferably, the pH value of the MES buffer in step four is 5.8-6.2, and the raw materials for preparing the MES buffer include 2-(N-morpholino)ethanesulfonic acid monohydrate and ultrapure water, with a concentration of 0.09-0.11 mol / L.

[0017] Preferably, in step four, the washing process uses an ultrafiltration tube with a molecular weight cutoff of 100 kDa, an ultrafiltration speed of 4000-6000 g / min, an ultrafiltration time of 8-12 min each time, and repeats the ultrafiltration 3 times.

[0018] Preferably, in step five, the concentration of the DMSO solution of rolipran is 4.8-5.2 mg / mL, and the concentration of the DMSO solution of 4-dimethylaminopyridine is 0.32-0.36 mg / mL.

[0019] Preferably, the washing of the reaction product in step five is performed using gradient centrifugation: the first centrifugation speed is 8000-10000 g / min, and the centrifugation time is 10-12 min;

[0020] The second centrifugation was performed at a speed of 12,000-14,000 g / min for 8-10 min; the third centrifugation was performed at a speed of 15,000-17,000 g / min for 5-7 min. The supernatant was discarded after each centrifugation, and the precipitate was resuspended in ultrapure water.

[0021] Preferably, the transaxonal retrograde nanomedicine based on gold nanoparticles obtained in step five is subjected to freeze-drying treatment, and the freeze-drying parameters are: pre-freezing temperature -40±2℃, pre-freezing time 3-4h;

[0022] The sublimation stage is carried out at a temperature of -20±2℃, a vacuum of 10-15Pa, and a time of 12-15h; the desorption stage is carried out at a temperature of 25±2℃, a vacuum of 5-8Pa, and a time of 6-8h.

[0023] Compared with existing technologies, this invention provides a method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles, which has the following beneficial effects:

[0024] 1. In this invention, during the ligand modification step for preparing gold nanoparticles, a stable carboxylation modification layer is formed on the surface of the gold nanoparticles by surface modification with mercaptosuccinic acid in an alkaline pH environment. This enhances the colloidal stability of the nanoparticles, prevents them from agglomerating in the physiological environment, and provides active sites for subsequent coupling of functional molecules.

[0025] 2. In this invention, in the coupling step of cholera toxin B subunit and gold nanoparticles, the GM1 ganglioside receptor binding activity of CTB is completely preserved by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and n-hydroxysuccinamide to catalyze the covalent coupling reaction, enabling it to recognize neuronal surface receptors and achieve efficient transaxonal retrograde transport of nanomedicines.

[0026] 3. In this invention, during the drug loading and purification steps, 4-dimethylaminopyridine catalyzes the esterification reaction of rolipran with modified gold nanoparticles, combined with gradient centrifugation to remove free drug and aggregates. This improves the drug loading rate while ensuring the uniformity of nanoparticle size, thereby obtaining high-purity, high-activity transaxonal retrograde nanomedicine. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: A method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles, comprising the following steps:

[0029] Step 1: Pretreatment of the vessels. Take a round-bottom flask, glass stopper and magnetic stir bar and immerse them in aqua regia solution for 25 minutes. After taking them out, rinse them three times with ultrapure water and place them in a 65℃ electric heating drying oven to dry for 2 hours.

[0030] Step 2: Synthesis of gold nanoparticles. Add 20 mL of an aqueous solution containing gold chloro-acid (HAuCl4) to a pretreated round-bottom flask. The concentration of HAuCl4 in the gold chloro-acid solution is 2.4 × 10⁻⁶. -4 The concentration of sodium tricitrate was 2.4 × 10 mol / L. -4 At a stirring rate of 800 r / min, 0.55 mL of a pre-cooled NaBH4 solution with a concentration of 0.09 mol / L was added at once, and stirring was continued until the solution color changed from light yellow to wine red, thus obtaining the initial solution of gold nanoparticles.

[0031] Step 3: Ligand modification. The pH of the initial solution of gold nanoparticles was adjusted to 10.5 with 0.08 mol / L NaOH solution. Mercaptosuccinic acid was added, and the molar ratio of mercaptosuccinic acid to HAuCl4 was 1.8:1. The mixture was stirred at 400 r / min for 10 h at room temperature. Then it was washed with deionized water and ultracentrifuged to obtain a concentrated solution of gold nanoparticles with a concentration of 2.4 mg / mL.

[0032] Step 4: CTB coupling. 2.4 mg of cholera toxin B subunit, 4.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2.4 mg of n-hydroxysuccinimide were dissolved in 0.09 mol / L MES buffer and mixed with gold nanoparticle concentrate. The mixture was stirred at 200 r / min for 45 min at 2 °C. After the reaction was completed, the mixture was washed with ultrapure water at 2 °C and centrifuged at 10000 g / min to obtain the AuNP-CTB complex.

[0033] Step 5: Drug loading. The AuNP-CTB complex was diluted with PBS to 4.8 mg (Au) / mL, and 1 mL of DMSO solution containing 4.8 mg roliplan and 5 mL of DMSO solution containing 1.6 mL 4-dimethylaminopyridine were added. The mixture was stirred at 300 r / min for 38 h at 23 °C in the dark. The reaction product was washed with ultrapure water and collected to obtain a transaxonal retrograde nanomedicine based on gold nanoparticles.

[0034] In step one, the aqua regia solution is prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1, and the temperature of the aqua regia solution is controlled at 20℃.

[0035] The preparation method of the aqueous solution of chloroform in step two is as follows: Dissolve solid HAuCl4 in ultrapure water, ultrasonically disperse for 5 min, then add sodium tricitrate, and continue ultrasonic dispersion for 10 min. The ultrasonic power is 200W.

[0036] The method for preparing the NaBH4 pre-cooled solution in step two is as follows: dissolve NaBH4 solid in deionized water pre-cooled at 0℃, prepare and use immediately, and the time interval between preparation and addition of gold chloro acid aqueous solution shall not exceed 5 minutes.

[0037] The parameters for ultracentrifugation in step three are: rotation speed 12000g / min, centrifugation time 15min, centrifugation temperature 4℃, and repeat centrifugation twice.

[0038] In step four, the pH of the MES buffer is 5.8. The raw materials for preparing the MES buffer include 2-(N-morpholino)ethanesulfonic acid monohydrate and ultrapure water, with a concentration of 0.09 mol / L.

[0039] In step four, the washing process uses an ultrafiltration tube with a molecular weight cutoff of 100 kDa, an ultrafiltration speed of 4000 g / min, an ultrafiltration time of 8 min each time, and repeats the ultrafiltration 3 times.

[0040] In step five, the concentration of rolipran in the DMSO solution was 4.8 mg / mL, and the concentration of 4-dimethylaminopyridine in the DMSO solution was 0.32 mg / mL.

[0041] In step five, the reaction product is washed using gradient centrifugation: the first centrifugation speed is 8000 g / min, and the centrifugation time is 10 min;

[0042] The second centrifugation was performed at a speed of 12000 g / min for 8 min; the third centrifugation was performed at a speed of 15000 g / min for 5 min. The supernatant was discarded after each centrifugation, and the precipitate was resuspended in ultrapure water.

[0043] The transaxon retrograde nanomedicine based on gold nanoparticles obtained in step five was subjected to freeze-drying. The freeze-drying parameters were: pre-freezing temperature -38℃ and pre-freezing time 3h.

[0044] The sublimation stage was held at -18℃, with a vacuum of 10Pa, for 12 hours; the desorption stage was held at 23℃, with a vacuum of 5Pa, for 6 hours.

[0045] Example 2: A method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles, comprising the following steps:

[0046] Step 1: Pretreatment of the vessels. Take a round-bottom flask, glass stopper and magnetic stir bar and immerse them in aqua regia solution for 30 minutes. After taking them out, rinse them with ultrapure water 4 times in sequence, and place them in a 70℃ electric heating drying oven to dry for 2.5 hours.

[0047] Step 2: Synthesis of gold nanoparticles. Add 20 mL of an aqueous solution containing gold chloro-acid (HAuCl4) to a pretreated round-bottom flask. The concentration of HAuCl4 in the gold chloro-acid solution is 2.5 × 10⁻⁶. -4 The concentration of sodium tricitrate was 2.5 × 10 mol / L. -4 At a stirring rate of 1000 r / min, 0.60 mL of a pre-cooled NaBH4 solution with a concentration of 0.10 mol / L was added at once, and stirring was continued until the solution color changed from light yellow to wine red to obtain the initial solution of gold nanoparticles.

[0048] Step 3: Ligand modification. The pH of the initial solution of gold nanoparticles was adjusted to 11.0 with 0.10 mol / L NaOH solution. Mercaptosuccinic acid was added, and the molar ratio of mercaptosuccinic acid to HAuCl4 was 2.0:1. The mixture was stirred at 500 r / min for 12 h at room temperature. Then it was washed with deionized water and ultracentrifuged to obtain a concentrated solution of gold nanoparticles with a concentration of 2.5 mg / mL.

[0049] Step 4: CTB coupling. 2.5 mg of cholera toxin B subunit, 5.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2.5 mg of n-hydroxysuccinimide were dissolved in 0.10 mol / L MES buffer and mixed with gold nanoparticle concentrate. The mixture was stirred at 300 r / min for 60 min at 4 °C. After the reaction was completed, the mixture was washed with ultrapure water at 4 °C and centrifuged at 11000 g / min to obtain the AuNP-CTB complex.

[0050] Step 5: Drug loading. The AuNP-CTB complex was diluted with PBS to 5.0 mg (Au) / mL, and 1 mL of DMSO solution containing 5.0 mg roliplan and 5 mL of DMSO solution containing 1.7 mL 4-dimethylaminopyridine were added. The mixture was stirred at 400 r / min for 40 h at 25 °C in the dark. The reaction product was washed with ultrapure water and collected to obtain a transaxonal retrograde nanomedicine based on gold nanoparticles.

[0051] In step one, the aqua regia solution is prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1, and the temperature of the aqua regia solution is controlled at 22℃.

[0052] The preparation method of the aqueous solution of chloroform in step two is as follows: Dissolve solid HAuCl4 in ultrapure water, ultrasonically disperse for 7 min, then add sodium tricitrate, and continue ultrasonic dispersion for 12 min. The ultrasonic power is 250W.

[0053] The method for preparing the NaBH4 pre-cooled solution in step two is as follows: dissolve NaBH4 solid in deionized water pre-cooled at 2℃, prepare and use immediately, and the time interval between preparation and addition of gold chloro acid aqueous solution shall not exceed 5 minutes.

[0054] The parameters for ultracentrifugation in step three are: rotation speed 13500g / min, centrifugation time 17min, centrifugation temperature 6℃, and repeat centrifugation twice.

[0055] In step four, the pH of the MES buffer is 6.0. The raw materials for preparing the MES buffer include 2-(N-morpholino)ethanesulfonic acid monohydrate and ultrapure water, with a concentration of 0.10 mol / L.

[0056] In step four, the washing process uses an ultrafiltration tube with a molecular weight cutoff of 100 kDa, an ultrafiltration speed of 5000 g / min, an ultrafiltration time of 10 min each time, and repeats the ultrafiltration 3 times.

[0057] In step five, the concentration of rolipran in the DMSO solution is 5.0 mg / mL, and the concentration of 4-dimethylaminopyridine in the DMSO solution is 0.34 mg / mL.

[0058] In step five, the reaction product is washed using gradient centrifugation: the first centrifugation speed is 9000 g / min, and the centrifugation time is 11 min;

[0059] The second centrifugation was performed at a speed of 13000 g / min for 9 min; the third centrifugation was performed at a speed of 16000 g / min for 3 min. The supernatant was discarded after each centrifugation, and the precipitate was resuspended in ultrapure water.

[0060] The gold nanoparticle-based transaxon retrograde nanomedicine obtained in step five was freeze-dried. The freeze-drying parameters were: pre-freezing temperature -40℃ and pre-freezing time 3.5h.

[0061] The sublimation stage was held at -20℃, with a vacuum of 12Pa, for 13 hours; the desorption stage was held at 25℃, with a vacuum of 6Pa, for 7 hours.

[0062] Example 3: A method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles, comprising the following steps:

[0063] Step 1: Pretreatment of the vessels. Take a round-bottom flask, glass stopper and magnetic stir bar and immerse them in aqua regia solution for 35 minutes. After taking them out, rinse them with ultrapure water 5 times in sequence and place them in a 75℃ electric heating drying oven to dry for 3 hours.

[0064] Step 2: Synthesis of gold nanoparticles. Add 20 mL of an aqueous solution containing gold chloro-acid (HAuCl4) to a pretreated round-bottom flask. The concentration of HAuCl4 in the gold chloro-acid solution is 2.6 × 10⁻⁶. -4 The concentration of sodium tricitrate was 2.6 × 10 mol / L. -4 At a stirring rate of 1200 r / min, 0.65 mL of a pre-cooled NaBH4 solution with a concentration of 0.11 mol / L was added at once, and stirring was continued until the solution color changed from light yellow to wine red, thus obtaining the initial solution of gold nanoparticles.

[0065] Step 3: Ligand modification. The pH of the initial solution of gold nanoparticles was adjusted to 11.5 with 0.12 mol / L NaOH solution. Mercaptosuccinic acid was added, and the molar ratio of mercaptosuccinic acid to HAuCl4 was 2.2:1. The mixture was stirred at 600 r / min for 14 h at room temperature. Then it was washed with deionized water and ultracentrifuged to obtain a concentrated solution of gold nanoparticles with a concentration of 2.6 mg / mL.

[0066] Step 4: CTB coupling. 2.6 mg of cholera toxin B subunit, 5.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2.6 mg of n-hydroxysuccinimide were dissolved in 0.11 mol / L MES buffer and mixed with gold nanoparticle concentrate. The mixture was stirred at 400 r / min for 75 min at 6 °C. After the reaction was completed, the mixture was washed with ultrapure water at 6 °C and centrifuged at 12000 g / min to obtain the AuNP-CTB complex.

[0067] Step 5: Drug loading. The AuNP-CTB complex was diluted with PBS to 5.2 mg (Au) / mL, and 1 mL of DMSO solution containing 5.2 mg roliplan and 5 mL of DMSO solution containing 1.8 mL 4-dimethylaminopyridine were added. The mixture was stirred at 500 r / min for 38-42 h at 27 °C in the dark. The reaction product was washed with ultrapure water and collected to obtain the transaxonal retrograde nanomedicine based on gold nanoparticles.

[0068] In step one, the aqua regia solution is prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1, and the temperature of the aqua regia solution is controlled at 25℃.

[0069] The preparation method of the aqueous solution of chloroform in step two is as follows: Dissolve solid HAuCl4 in ultrapure water, ultrasonically disperse for 10 min, then add sodium tricitrate, and continue ultrasonic dispersion for 15 min. The ultrasonic power is 300W.

[0070] The method for preparing the NaBH4 pre-cooled solution in step two is as follows: dissolve NaBH4 solid in deionized water pre-cooled at 4℃, prepare and use immediately, and the time interval between preparation and addition of gold chloro acid aqueous solution shall not exceed 5 minutes.

[0071] The parameters for ultracentrifugation in step three are: rotation speed 15000g / min, centrifugation time 20min, centrifugation temperature 8℃, and repeat centrifugation 3 times.

[0072] In step four, the pH of the MES buffer is 6.2. The raw materials for preparing the MES buffer include 2-(N-morpholino)ethanesulfonic acid monohydrate and ultrapure water, with a concentration of 0.11 mol / L.

[0073] In step four, the washing process uses an ultrafiltration tube with a molecular weight cutoff of 100 kDa, an ultrafiltration speed of 6000 g / min, an ultrafiltration time of 12 min each time, and repeats the ultrafiltration 3 times.

[0074] In step five, the concentration of rolipran in the DMSO solution was 5.2 mg / mL, and the concentration of 4-dimethylaminopyridine in the DMSO solution was 0.36 mg / mL.

[0075] In step five, the reaction product is washed using gradient centrifugation: the first centrifugation speed is 10000 g / min, and the centrifugation time is 12 min;

[0076] The second centrifugation was performed at a speed of 14,000 g / min for 10 min; the third centrifugation was performed at a speed of 17,000 g / min for 7 min. The supernatant was discarded after each centrifugation, and the precipitate was resuspended in ultrapure water.

[0077] The transaxon retrograde nanomedicine based on gold nanoparticles obtained in step five was subjected to freeze-drying. The freeze-drying parameters were: pre-freezing temperature -42℃ and pre-freezing time 4h.

[0078] The sublimation stage was conducted at a temperature of -22℃, a vacuum of 15Pa, and a time of 15 hours; the desorption stage was conducted at a temperature of 27℃, a vacuum of 8Pa, and a time of 8 hours.

[0079] Comparative Example 1 differs from Example 1 in that: in step three, during ligand modification, NaOH solution was not used to adjust the initial pH of the gold nanoparticle solution, and the initial pH was maintained for subsequent reactions.

[0080] Comparative Example 2 differs from Example 1 in that: in step four, CTB coupling in this comparative example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and n-hydroxysuccinamide were not added; only the cholera toxin B subunit was mixed with the gold nanoparticle concentrate.

[0081] Comparative Example 3 differs from Example 1 in that: in step five, 4-dimethylaminopyridine was not added during drug loading in this comparative example; instead, the DMSO solution of rolipran was mixed with the AuNP-CTB complex for reaction.

[0082] Comparative Example 4 differs from Example 1 in that: in step five, when washing the reaction product, gradient centrifugation was not used in this comparative example; instead, a single centrifugation at 12000g / min for 15min was performed to collect the precipitate.

[0083] The performance of the gold nanoparticle-based transaxonal retrograde nanomedicines prepared in Examples 1-3 and Comparative Examples 1-4 was tested. The test items and methods are as follows:

[0084] Particle size and dispersibility were tested using a dynamic light scattering instrument to detect the hydrated particle size and polydispersity index of the nanoparticles at a test temperature of 25°C and an equilibration time of 120s.

[0085] Zeta potential testing was performed using electrophoretic light scattering to measure the zeta potential on the surface of nanoparticles under the same instrument to evaluate colloidal stability.

[0086] The cross-axon retrograde efficiency test involved co-incubating nanomedicines with in vitro cultured dorsal root ganglion neurons for 12 hours. After fixation, CTB and axonal markers were labeled by immunofluorescence staining, and the percentage of nanoparticles that entered the axon and retrograded to the cell body was counted.

[0087] Drug loading rate was tested by using high performance liquid chromatography to determine the concentration of rolipran in the supernatant before and after the reaction, and the loading was calculated as follows: drug loading = (initial drug load - free drug load) / total mass of nanoparticles × 100%.

[0088] The test data of the nanomedicines prepared in Examples 1-3 and Comparative Examples 1-4 are recorded in the table below:

[0089] Group Hydrated particle size (nm) polydispersion coefficient Zeta potential (mV) Transaxon retrograde efficiency (%) Drug loading rate (%) Example 1 28.5±1.2 0.12±0.02 -32.6±1.5 78.3±3.2 22.5±1.1 Example 2 26.8±0.9 0.10±0.01 -35.2±1.8 85.6±2.8 25.3±0.9 Example 3 27.3±1.1 0.11±0.02 -33.9±1.6 82.1±3.0 24.1±1.0 Comparative Example 1 45.7±2.5 0.28±0.05 -18.4±2.3 42.5±4.1 15.8±1.5 Comparative Example 2 32.1±1.8 0.19±0.03 -25.7±2.0 31.2±3.5 20.6±1.2 Comparative Example 3 29.6±1.3 0.13±0.02 -30.8±1.7 76.8±3.3 12.4±0.8 Comparative Example 4 38.9±2.0 0.22±0.04 -28.3±1.9 65.4±3.8 18.7±1.3

[0090] By comparing and analyzing the data in the table, it can be seen that the transaxonal retrograde nanomedicines based on gold nanoparticles prepared by the processes in Examples 1-3 have significantly better performance than the transaxonal retrograde nanomedicines prepared by the processes in Comparative Examples 1-4. This indicates that the present invention, through surface modification with mercaptosuccinic acid in an alkaline pH environment, forms a stable carboxylation modification layer on the surface of gold nanoparticles, improves the colloidal stability of the nanoparticles, avoids their aggregation in the physiological environment, and provides active sites for subsequent coupling of functional molecules; by using 1-ethyl-3 The covalent coupling reaction catalyzed by 3-dimethylaminopropyl)carbodiimide and n-hydroxysuccinamide fully preserves the GM1 ganglioside receptor binding activity of CTB, enabling it to recognize neuronal surface receptors and achieve efficient transaxonal retrograde transport of nanomedicines. The esterification reaction of rolipran with modified gold nanoparticles catalyzed by 4-dimethylaminopyridine, combined with gradient centrifugation to remove free drug and aggregates, improves drug loading while ensuring nanoparticle size uniformity, thus obtaining high-purity, highly active transaxonal retrograde nanomedicines.

[0091] By comparing and analyzing the relevant data in the table, it can be seen that the gold nanoparticle-based transaxonal retrograde nanomedicine prepared by the method of the present invention has good particle size uniformity, colloidal stability, transaxonal retrograde efficiency and drug loading rate.

[0092] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing transaxonal retrograde nanomedicines based on gold nanoparticles, characterized in that: Includes the following steps: Step 1: Pretreatment of the vessels. Immerse the round-bottom flask, glass stopper and magnetic stir bar in aqua regia solution for 25-35 minutes. After taking them out, rinse them with ultrapure water 3-5 times in sequence, and place them in an electric heating drying oven at 70±5℃ for 2-3 hours to dry. Step 2: Synthesis of gold nanoparticles. Add 20 mL of an aqueous solution containing gold chloro acid to a pretreated round-bottom flask. The concentration of HAuCl4 in the gold chloro acid aqueous solution is 2.4 × 10⁻⁶. -4 -2.6×10 -4 The concentration of sodium tricitrate was 2.4 × 10 mol / L. -4 -2.6×10 -4 At a stirring rate of 800-1200 r / min, 0.55-0.65 mL of a pre-cooled NaBH4 solution with a concentration of 0.09-0.11 mol / L was added at once, and stirring was continued until the solution color changed from light yellow to wine red to obtain the initial solution of gold nanoparticles. Step 3: Ligand modification. The pH of the initial gold nanoparticle solution was adjusted to 10.5-11.5 with 0.08-0.12 mol / L NaOH solution. Mercaptosuccinic acid was added, with a molar ratio of mercaptosuccinic acid to HAuCl4 of 1.8-2.2:

1. The mixture was stirred at 400-600 r / min for 10-14 h at room temperature. The solution was then washed with deionized water and subjected to ultracentrifugation to obtain a concentrated gold nanoparticle solution with a concentration of 2.4-2.6 mg / mL. Step 4: CTB coupling. Take 2.4-2.6 mg of cholera toxin B subunit, 4.8-5.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2.4-2.6 mg of n-hydroxysuccinimide and dissolve them in 0.09-0.11 mol / L MES buffer. Mix with gold nanoparticle concentrate and stir at 200-400 r / min for 45-75 min at 4±2℃. After the reaction is completed, wash with ultrapure water at 4±2℃ and centrifuge at 10000-12000 g / min to obtain AuNP-CTB complex. Step 5: Drug loading. Dilute the AuNP-CTB complex with PBS to 4.8-5.2 mg (Au) / mL, add 1 mL of DMSO solution containing 4.8-5.2 mg rolipran and 5 mL of DMSO solution containing 1.6-1.8 mL 4-dimethylaminopyridine, and stir at 300-500 r / min for 38-42 h at 25±2℃ in the dark. The reaction product is washed with ultrapure water and collected to obtain the transaxonal retrograde nanomedicine based on gold nanoparticles.

2. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: In step one, the aqua regia solution is prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1, and the temperature of the aqua regia solution is controlled at 20-25℃.

3. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: The preparation method of the aqueous solution of chloroform in step two is as follows: dissolve solid HAuCl4 in ultrapure water, ultrasonically disperse for 5-10 min, then add sodium tricitrate, and continue ultrasonic dispersion for 10-15 min, with an ultrasonic power of 200-300W.

4. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: The method for preparing the NaBH4 pre-cooled solution in step two is as follows: dissolve NaBH4 solid in deionized water pre-cooled at 0-4℃, prepare and use immediately, and the time interval from preparation to addition of gold chloro acid aqueous solution shall not exceed 5 minutes.

5. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: The parameters for ultracentrifugation in step three are: rotation speed 12000-15000 g / min, centrifugation time 15-20 min, centrifugation temperature 4-8℃, and repeated centrifugation 2-3 times.

6. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: In step four, the pH value of the MES buffer is 5.8-6.2, and the raw materials for preparing the MES buffer include 2-(N-morpholino)ethanesulfonic acid monohydrate and ultrapure water, with a concentration of 0.09-0.11 mol / L.

7. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: In step four, the washing process uses an ultrafiltration tube with a molecular weight cutoff of 100 kDa, an ultrafiltration speed of 4000-6000 g / min, an ultrafiltration time of 8-12 min each time, and repeats the ultrafiltration 3 times.

8. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: In step five, the concentration of rolipran in the DMSO solution is 4.8-5.2 mg / mL, and the concentration of 4-dimethylaminopyridine in the DMSO solution is 0.32-0.36 mg / mL.

9. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: In step five, the reaction product is washed using a gradient centrifugation method: the first centrifugation speed is 8000-10000 g / min, and the centrifugation time is 10-12 min; The second centrifugation was performed at a speed of 12000-14000 g / min for 8-10 min. The third centrifugation was performed at a speed of 15,000-17,000 g / min for 5-7 min. The supernatant was discarded after each centrifugation, and the precipitate was resuspended in ultrapure water.

10. The method for preparing transaxonal retrograde nanomedicine based on gold nanoparticles according to claim 1, characterized in that: The gold nanoparticle-based transaxon retrograde nanomedicine obtained in step five was subjected to freeze-drying. The freeze-drying parameters were: pre-freezing temperature -40±2℃, pre-freezing time 3-4h. The sublimation stage is carried out at a temperature of -20±2℃, a vacuum of 10-15Pa, and a time of 12-15h; the desorption stage is carried out at a temperature of 25±2℃, a vacuum of 5-8Pa, and a time of 6-8h.