Preparation method of drug delivery nanoparticles based on hydrogel microneedle

Abstract

The invention discloses a preparation method of drug delivery nanoparticles based on hydrogel microneedles, and relates to the technical field of biological medicine, the technical key points are as follows: step S1, preparing a white suspension solution; step S2, preparing a white powdery porous hollow nano microsphere A; step S3, preparing A nano microsphere powder loaded with TP; step S4, preparing a CaCO3 (at) Lip (at) DEC205 nano microsphere preparation; and S5, preparing the PVP microneedle: dropwise adding a proper amount of polyvinylpyrrolidone solution into the mold, keeping the weights of the two six-hole culture plates equal, centrifuging, putting the obtained culture plates into a drying oven, evaporating the solution to dryness, and taking down the prepared microneedle from the mold. The liposome is added, so that the TP drug loading efficiency is higher, and the toxic and side effects of the whole body are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing hydrogel microneedle-based drug delivery nanoparticles. Background Art

[0002] Transplant rejection is an insurmountable problem faced after organ transplantation. The anti-rejection drugs currently used can cause systemic toxic side effects. Severe postoperative infection and the occurrence of metabolic syndrome have become important factors for postoperative death of organ transplant patients. At the same time, tumor recurrence caused by immunosuppression has also become a thorny problem.

[0003] Triptolide (TP) has a strong immunosuppressive function and has achieved good results in inducing immune tolerance in organ transplantation. TP also has anti-tumor efficacy, but its clinical application is limited due to its narrow therapeutic window and high toxicity risk. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a method for preparing drug delivery nanoparticles based on hydrogel microneedles.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention provides a method for preparing drug delivery nanoparticles based on hydrogel microneedles, comprising the following steps:

[0007] Step S1, at room temperature, measure CaCl2 solution in a 1000ml beaker, then measure 200ml SDS solution, and quickly mix under stirring. After the solution changes from clear to milky white, continue to stir and add 400ml Na2CO3 solution, and the solution begins to become a white suspension solution;

[0008] Step S2, sealing the white suspension solution with a sealing film in a magnetic stirrer (1600 r / min), and washing with deionized water and anhydrous ethanol for 3 times respectively, collecting the precipitate and drying it in a 60° C. oven for 1 h, and obtaining white powdery porous hollow nanospheres A after drying;

[0009] Step S3, weighing the TP standard and placing it in a 100 ml conical flask, adding 20 ml of methanol solution to completely dissolve it, then accurately weighing 0.3 g of A microsphere powder and placing it in the conical flask, sealing the conical flask mouth with a sealing film, placing it in a 37°C desktop constant temperature oscillating box and oscillating at a rate of 150 r / min, and measuring the absorbance value of TP every 2 hours; when its absorbance value is almost stable, the loading amount of TP has reached saturation, and the oscillation is stopped at this time;

[0010] Step S4, collecting the precipitate of the TP-loaded A nanosphere powder by centrifugation (5500 r / min), repeatedly washing the precipitate with dilute ammonia water with pH = 7.4 and deionized water to remove the TP adsorbed on the surface of the TP / CaCO3 nanosphere, and then combining it with the antibody Anti-DEC205 to obtain the CaCO3@Lip@DEC205 nanosphere preparation;

[0011] Step S5, preparation of PVP microneedles: prepare polyvinyl pyrrolidone solution, take polyvinyl pyrrolidone in a glass reagent bottle, add deionized water, heat and stir until polyvinyl pyrrolidone is fully dissolved; place an equal amount of PDMS molds in a six-well culture plate, drop an appropriate amount of polyvinyl pyrrolidone solution into the mold and keep the weight of the two six-well culture plates equal, select a speed of 4000 revolutions per minute for 5 minutes of centrifugation, take out the two six-well culture plates, add a few drops of solution with a rubber-tipped dropper, continue to keep the weight of the two six-well culture plates equal, centrifuge again for 5 minutes, repeat the operation 4 times; place the obtained culture plate in an oven set to 35 degrees Celsius for about 12 hours until the solution evaporates, and then the prepared microneedles can be removed from the mold.

[0012] The present invention is further configured as follows: in step S1, the CaCl2 solution is 0.1-0.5 mol / L, the SDS solution is 0.1 mol / L, and the Na2CO3 solution is 0.1-0.5 mol / L.

[0013] The present invention is further configured as follows: in step S3, the mass of the TP standard is 0.01-0.1 g.

[0014] The present invention is further configured as follows: the mass fraction of the polyvinyl pyrrolidone solution prepared in step S5 is 5-15%, the polyvinyl pyrrolidone is 10.0 g, and the deionized water is 100 mL.

[0015] Compared with the prior art, the beneficial effects of this solution are as follows: calcium carbonate biominerals have unique advantages as targeted carriers. They have ideal biocompatibility and biodegradability, are non-toxic to the human body, are easily excreted from the body, and have a large specific surface area and the ability to load various drugs, making them ideal drug carriers. Targeted delivery and release of drugs are achieved through endocytosis. TP is a fat-soluble drug, and the effect of calcium carbonate alone on drug delivery is not ideal. The addition of liposomes in the present invention makes TP more efficient. At the same time, the surface of the nanoparticles is connected to the monoclonal antibody Anti-DEC205 targeting DCs, so that the nanoparticles entering the body can target TP and deliver it to DCs to exert its effect, thereby reducing systemic toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a macroscopic schematic diagram of the calcium carbonate liposome nanoparticle microneedle material in an embodiment of the present invention;

[0017] Figure 2 It is the degree of rejection reaction of the blank group, the TP group, the TP-encapsulated nanoparticle group, and the TP-encapsulated nanoparticle group delivered to DC cells in the acute rejection reaction model of mouse heart transplantation in the embodiment of the present invention;

[0018] Figure 3 It is the expression amount of CD40 molecules in the spleen of the blank group, the TP group, the TP-encapsulated nanoparticle group, and the TP-encapsulated nanoparticle group delivered to DC cells in the mouse heart transplant acute rejection model in the embodiment of the present invention;

[0019] Figure 4 It is the expression amount of CD80 molecules in the spleen of the blank group, the TP group, the TP-encapsulated nanoparticle group, and the TP-encapsulated nanoparticle group targeted to DC cells in the mouse heart transplant acute rejection model in the embodiment of the present invention;

[0020] Figure 5 It is the expression amount of CD86 molecule in spleen of blank group, TP group, TP-encapsulated nanoparticle group, and TP-encapsulated nanoparticle group delivered to DC cells in the acute rejection reaction model of mouse heart transplantation in the embodiment of the present invention;

[0021] Figure 6 The hepatorenal toxicity hematological index detection of the blank group, TP group, TP-encapsulated nanoparticle group, and TP-encapsulated nanoparticle group delivered to DC cells in the acute rejection reaction model of mouse heart transplantation in the embodiment of the present invention;

[0022] Figure 7 It is the HE staining detection of other organ toxicity pathology in the mouse heart transplant acute rejection reaction model in the embodiment of the present invention, among the blank group, TP group, TP-encapsulated nanoparticle group, and TP-encapsulated nanoparticle group targeted to DC cells. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Embodiment 1:

[0026] Step 1: At room temperature, measure CaCl2 (0.2 mol / L) solution in a 1000 ml beaker, then measure 200 ml SDS (0.1 mol / L) solution, and mix quickly under stirring. After the solution changes from clear to milky white, continue to stir and add 400 ml Na2CO3 (0.2 mol / L) solution. The solution begins to become a white suspension solution.

[0027] Step 2: The prepared solution was sealed with a sealing film in a magnetic stirrer (1600 r / min), and washed with deionized water and anhydrous ethanol for 3 times respectively, and the precipitate was collected and placed in a 60° C. oven for drying for 1 h to obtain white powdery porous hollow nanospheres A;

[0028] Step 3: Accurately weigh 0.02g of TP standard and place it in a 100ml conical flask, add 20ml of methanol solution to completely dissolve it, then accurately weigh 0.3g of A microsphere powder and place it in the conical flask, seal the conical flask mouth with a sealing film, place it in a 37℃ desktop constant temperature oscillating box and oscillate at a rate of 150r / min, and measure the absorbance value of TP every 2h. When its absorbance value is almost stable, the loading amount of TP has reached saturation, and the oscillation is stopped at this time;

[0029] Step 4: The TP-loaded A nanosphere powder was centrifuged to collect the precipitate (5500 r / min), and the precipitate was repeatedly washed with dilute ammonia water with pH = 7.4 and deionized water to remove the TP adsorbed on the surface of the TP / CaCO3 nanospheres, and then combined with the antibody Anti-DEC205 to obtain the CaCO3@Lip@DEC205 nanosphere preparation;

[0030] Step 5: Prepare a 10% polyvinyl pyrrolidone solution by placing polyvinyl pyrrolidone in a glass reagent bottle, adding deionized water, and heating and stirring until the polyvinyl pyrrolidone is fully dissolved;

[0031] Step 6: Place an equal amount of PDMS molds in the six-well culture plates, drip an appropriate amount of 10% polyvinyl pyrrolidone solution into the molds and keep the weight of the two six-well culture plates equal, select a speed of 4000 revolutions per minute for 5 minutes of centrifugation, take out the two six-well culture plates, add a few drops of solution with a rubber-tipped dropper, continue to keep the weight of the two six-well culture plates equal, centrifuge again for 5 minutes, repeat the operation 4 times; place the resulting culture plates in an oven set to 35 degrees Celsius for about 12 hours until the solution evaporates, and then the prepared microneedles can be removed from the molds.

[0032] Embodiment 2:

[0033] Step 1: At room temperature, measure CaCl2 (0.4 mol / L) solution in a 1000 ml beaker, then measure 200 ml SDS (0.1 mol / L) solution, and mix quickly under stirring. After the solution changes from clear to milky white, continue to stir and add 400 ml Na2CO3 (0.4 mol / L) solution. The solution begins to become a white suspension solution.

[0034] Step 2: The prepared solution was sealed with a sealing film in a magnetic stirrer (1600 r / min), and washed with deionized water and anhydrous ethanol for 3 times respectively, and the precipitate was collected and placed in a 60° C. oven for drying for 1 h to obtain white powdery porous hollow nanospheres A;

[0035] Step 3: Accurately weigh 0.04g of TP standard and place it in a 100ml conical flask, add 20ml of methanol solution to completely dissolve it, then accurately weigh 0.3g of A microsphere powder and place it in the conical flask, seal the conical flask mouth with a sealing film, place it in a 37℃ desktop constant temperature oscillating box and oscillate at a rate of 150r / min, and measure the absorbance value of TP every 2h. When its absorbance value is almost stable, the loading amount of TP has reached saturation, and the oscillation is stopped at this time;

[0036] Step 4: The TP-loaded A nanosphere powder was centrifuged to collect the precipitate (5500 r / min), and the precipitate was repeatedly washed with dilute ammonia water with pH = 7.4 and deionized water to remove the TP adsorbed on the surface of the TP / CaCO3 nanospheres, and then combined with the antibody Anti-DEC205 to obtain the CaCO3@Lip@DEC205 nanosphere preparation;

[0037] Step 5: Prepare 8% polyvinyl pyrrolidone solution, take polyvinyl pyrrolidone into a glass reagent bottle, add deionized water, heat and stir until the polyvinyl pyrrolidone is fully dissolved;

[0038] Step 6: Place an equal amount of PDMS molds in the six-well culture plates, drip an appropriate amount of 10% polyvinyl pyrrolidone solution into the molds and keep the weight of the two six-well culture plates equal, select a speed of 4000 revolutions per minute for 5 minutes of centrifugation, take out the two six-well culture plates, add a few drops of solution with a rubber-tipped dropper, continue to keep the weight of the two six-well culture plates equal, centrifuge again for 5 minutes, repeat the operation 4 times; place the resulting culture plates in an oven set to 35 degrees Celsius for about 12 hours until the solution evaporates, and then the prepared microneedles can be removed from the molds.

[0039] Embodiment 3:

[0040] Step 1: At room temperature, measure CaCl2 (0.2 mol / L) solution in a 1000 ml beaker, then measure 200 ml SDS (0.1 mol / L) solution, and mix quickly under stirring. After the solution changes from clear to milky white, continue to stir and add 400 ml Na2CO3 (0.2 mol / L) solution. The solution begins to become a white suspension solution.

[0041] Step 2: The prepared solution was sealed with a sealing film in a magnetic stirrer (1600 r / min), and washed with deionized water and anhydrous ethanol for 3 times respectively, and the precipitate was collected and placed in a 60° C. oven for drying for 1 h to obtain white powdery porous hollow nanospheres A;

[0042] Step 3: Accurately weigh 0.02g of TP standard and place it in a 100ml conical flask, add 20ml of methanol solution to completely dissolve it, then accurately weigh 0.3g of A microsphere powder and place it in the conical flask, seal the conical flask mouth with a sealing film, place it in a 37℃ desktop constant temperature oscillating box and oscillate at a rate of 150r / min, and measure the absorbance value of TP every 2h. When its absorbance value is almost stable, the loading amount of TP has reached saturation, and the oscillation is stopped at this time;

[0043] Step 4: The TP-loaded A nanosphere powder was centrifuged to collect the precipitate (5500 r / min), and the precipitate was repeatedly washed with dilute ammonia water with pH = 7.4 and deionized water to remove the TP adsorbed on the surface of the TP / CaCO3 nanospheres, and then combined with the antibody Anti-DEC205 to obtain the CaCO3@Lip@DEC205 nanosphere preparation;

[0044] Step 5: Prepare a 10% polyvinyl pyrrolidone solution by placing polyvinyl pyrrolidone in a glass reagent bottle, adding deionized water, and heating and stirring until the polyvinyl pyrrolidone is fully dissolved;

[0045] Step 6: Place an equal amount of PDMS molds in the six-well culture plates, drip an appropriate amount of 12% polyvinyl pyrrolidone solution into the molds and keep the weight of the two six-well culture plates equal, select a speed of 4000 revolutions per minute for 5 minutes of centrifugation, take out the two six-well culture plates, add a few drops of solution with a rubber-tipped dropper, continue to keep the weight of the two six-well culture plates equal, centrifuge again for 5 minutes, and repeat the operation 4 times; place the resulting culture plates in an oven set to 35 degrees Celsius for about 12 hours until the solution evaporates, and then the prepared microneedles can be removed from the molds.

[0046] TP is a fat-soluble drug. Calcium carbonate liposome nanoparticles can carry TP to the maximum extent. Antibodies targeting DC are carried on the surface of the particles and accurately released around DC to exert their effects. HE staining results show that in the acute rejection model of mouse heart transplantation, the degree of rejection of the group of TP nanoparticles targeted for delivery to DC cells was reduced compared with the blank group, TP group, and TP nanoparticles group. The levels of CD40, CD80, and CD86 molecules in the spleen were reduced, indicating that the group of TP nanoparticles targeted for delivery to DC cells can reduce the rejection reaction after heart transplantation. In terms of toxicity detection, it can be seen that the TP-treated group has greater liver and kidney toxicity ( Figure 6 ), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher than those in the nanoparticle group. In terms of renal toxicity, serum creatinine (Cr) and urea nitrogen (Urea) in the TP systemic medication group were significantly higher than those in the nanoparticle group. Pathological HE staining showed that the toxicity of the liver, kidney, lung, and spleen in the TP group was higher than that in the nanoparticle group.

[0047] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing drug delivery nanoparticles based on hydrogel microneedles, characterized in that: The following steps are involved: Step S1, at room temperature, measure CaCl2 solution into a 1000ml beaker, then measure 200ml SDS solution, and quickly mix under stirring. After the solution changes from clear to milky white, continue to stir and add 400ml Na2CO3 solution, and the solution begins to change into a white suspension solution; Step S2, sealing the white suspension solution with a sealing film in a magnetic stirrer (1600 r / min), and washing with deionized water and anhydrous ethanol for 3 times respectively, collecting the precipitate and drying it in a 60° C. oven for 1 h, and obtaining white powdery porous hollow nanospheres A after drying; Step S3, weigh the TP standard and place it in a 100 ml conical flask, add 20 ml of methanol solution to completely dissolve it, then accurately weigh 0.3 g of A microsphere powder and place it in the conical flask, seal the conical flask mouth with a sealing film, place it in a 37 ° C desktop constant temperature oscillation box and oscillate at a rate of 150 r / min, and measure the absorbance value of TP every 2 hours; When the absorbance value is almost stable, the loading amount of TP has reached saturation, and the oscillation is stopped at this time; Step S4, collecting the precipitate of the TP-loaded A nanosphere powder by centrifugation (5500 r / min), repeatedly washing the precipitate with dilute ammonia water with pH = 7.4 and deionized water to remove the TP adsorbed on the surface of the TP / CaCO3 nanosphere, and then combining it with the antibody Anti-DEC205 to obtain the CaCO3@Lip@DEC205 nanosphere preparation; Step S5, preparation of PVP microneedles: prepare polyvinyl pyrrolidone solution, take polyvinyl pyrrolidone in a glass reagent bottle, add deionized water, heat and stir until polyvinyl pyrrolidone is fully dissolved; place an equal amount of PDMS molds in a six-well culture plate, drop an appropriate amount of polyvinyl pyrrolidone solution into the mold and keep the weight of the two six-well culture plates equal, select a speed of 4000 revolutions per minute for 5 minutes of centrifugation, take out the two six-well culture plates, add a few drops of solution with a rubber-tipped dropper, continue to keep the weight of the two six-well culture plates equal, centrifuge again for 5 minutes, repeat the operation 4 times; place the obtained culture plate in an oven set to 35 degrees Celsius for about 12 hours until the solution evaporates, and then the prepared microneedles can be removed from the mold.

2. A method for preparing drug delivery nanoparticles based on hydrogel microneedles as claimed in claim 1, characterized in that: In step S1, the CaCl2 solution is 0.1-0.5 mol / L, the SDS solution is 0.1 mol / L, and the Na2CO3 solution is 0.1-0.5 mol / L.

3. The method for preparing drug delivery nanoparticles based on hydrogel microneedles according to claim 1, characterized in that: The mass of the TP standard in step S3 is 0.01-0.1 g.

4. The method for preparing drug delivery nanoparticles based on hydrogel microneedles according to claim 1, characterized in that: The mass fraction of the polyvinyl pyrrolidone solution prepared in step S5 is 5-15%, the polyvinyl pyrrolidone is 10.0 g, and the deionized water is 100 mL.

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