CN113908287B - Preparation method of drug-loaded plant protein nano particles - Google Patents
Preparation method of drug-loaded plant protein nano particles Download PDFInfo
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- CN113908287B CN113908287B CN202111179912.6A CN202111179912A CN113908287B CN 113908287 B CN113908287 B CN 113908287B CN 202111179912 A CN202111179912 A CN 202111179912A CN 113908287 B CN113908287 B CN 113908287B
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- 239000003814 drug Substances 0.000 title claims abstract description 62
- 229940079593 drug Drugs 0.000 title claims abstract description 60
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 48
- 108010064851 Plant Proteins Proteins 0.000 title claims abstract description 22
- 235000021118 plant-derived protein Nutrition 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 89
- 108010082495 Dietary Plant Proteins Proteins 0.000 claims abstract description 36
- 239000007853 buffer solution Substances 0.000 claims abstract description 36
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 26
- 238000000502 dialysis Methods 0.000 claims abstract description 19
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 16
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 16
- 239000012498 ultrapure water Substances 0.000 claims abstract description 16
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 3
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 claims description 58
- 229920002494 Zein Polymers 0.000 claims description 47
- 229940093612 zein Drugs 0.000 claims description 47
- 239000005019 zein Substances 0.000 claims description 47
- 239000002245 particle Substances 0.000 claims description 41
- 235000012754 curcumin Nutrition 0.000 claims description 29
- 239000004148 curcumin Substances 0.000 claims description 29
- 229940109262 curcumin Drugs 0.000 claims description 29
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 claims description 29
- 239000012460 protein solution Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 claims description 8
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 claims description 8
- 239000000337 buffer salt Substances 0.000 claims description 8
- 235000012680 lutein Nutrition 0.000 claims description 8
- 229960005375 lutein Drugs 0.000 claims description 8
- 239000001656 lutein Substances 0.000 claims description 8
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 claims description 8
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 claims description 8
- 235000021283 resveratrol Nutrition 0.000 claims description 8
- 229940016667 resveratrol Drugs 0.000 claims description 8
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 claims description 8
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000012046 mixed solvent Substances 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 229940071440 soy protein isolate Drugs 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 239000012798 spherical particle Substances 0.000 abstract description 5
- 230000003321 amplification Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 53
- 239000000463 material Substances 0.000 description 38
- 239000000047 product Substances 0.000 description 26
- 238000009826 distribution Methods 0.000 description 18
- 235000019441 ethanol Nutrition 0.000 description 17
- 238000011068 loading method Methods 0.000 description 17
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 11
- 229910052708 sodium Inorganic materials 0.000 description 11
- 239000011734 sodium Substances 0.000 description 11
- 239000001509 sodium citrate Substances 0.000 description 11
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 11
- 238000005303 weighing Methods 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108010073771 Soybean Proteins Proteins 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 4
- 235000019799 monosodium phosphate Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
- 229940001941 soy protein Drugs 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001338 self-assembly Methods 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 239000012296 anti-solvent Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000019710 soybean protein Nutrition 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000011164 primary particle Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5052—Proteins, e.g. albumin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
Abstract
The invention discloses a preparation method of drug-loaded plant protein nano particles, which comprises the steps of dissolving plant protein and natural hydrophobic drugs to form a solution; preparing sodium citrate-citric acid or phosphate buffer; the solution of the vegetable protein and the natural hydrophobic drug is divided into two syringes and fixed on the same syringe pump; subpackaging the buffer solution in two syringes and fixing the syringes on the same syringe pump; introducing the solution in the four injectors into a multi-channel vortex mixer through an injection pump, and taking out the product at the outlet of the multi-channel vortex mixer; and (3) dialyzing and purifying the product in ultrapure water by using a dialysis bag to obtain the nano spherical particles with the vegetable protein as a carrier and the natural hydrophobic drug. The invention also discloses the plant protein drug-loaded nano spherical particles obtained by the method. The invention can realize continuous reaction and preparation of the nano spherical particles, and can effectively overcome the problems caused by intermittent preparation and amplification effects.
Description
Technical Field
The invention belongs to the technical field of functional nano materials, and particularly relates to a preparation method of drug-loaded plant protein nano particles.
Background
The natural plant protein has many advantages as drug-loaded nano particles, such as wide sources, excellent biocompatibility and biodegradability, etc., and zein and soy protein isolate are typical representatives thereof. The chemical structures of the two plant proteins simultaneously contain hydrophilic groups and hydrophobic groups to have amphipathy, so that the two plant proteins can self-assemble to form a nano structure and serve as a nano carrier.
At present, the relatively mature method for preparing the plant protein nanoparticles is an anti-solvent precipitation method and a pH driving method, which are based on self-assembly of thermodynamic equilibrium to form the nanoparticles. The anti-solvent precipitation method is to dissolve vegetable protein in a mixed solvent containing an organic solvent-water which is mutually soluble with water, and then add a large amount of water into the mixed solvent system to promote the self-assembly of the vegetable protein to form nano particles; finally, removing the organic solvent by means of centrifugation, evaporation or dialysis, etc., thereby obtaining the water-dispersible plant protein nano particles. The pH driving method is to dissolve vegetable protein in alkaline water solution, then to regulate the pH value of the original system to neutral with acid to promote the self-assembly of vegetable protein to form nanometer particle; and centrifuging to remove the precipitate in the system, wherein the obtained supernatant is the water-dispersible plant protein nanoparticle solution. However, both preparation methods have certain problems in material preparation and future industrial production:
1. the drug loading is low, and the grain size is difficult to control. In the traditional preparation method, in order to obtain the plant protein nano particles with stable structures, a large amount of plant proteins are needed to be added, the dosage is large, and a second polymer is needed to be added as a co-stabilizer, so that the drug loading rate of the prepared nano particles is low (usually less than 10%), and meanwhile, the particle size and the particle size distribution of the nano particles are difficult to systematically regulate;
2. the traditional preparation method is long in time consumption and batch-type, and is not easy to convert into large-scale continuous production.
Disclosure of Invention
The invention aims at overcoming the defects of the prior method for preparing the plant protein nano particles, thereby providing a novel preparation method, preparing stable drug-loaded nano particles without adding a second polymer, realizing the regulation and control of high drug-loaded quantity and particle size, and being beneficial to continuous mass production by using continuous preparation.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for preparing drug-loaded vegetable protein nanoparticles, comprising the steps of:
s1, dissolving vegetable protein in an organic solvent-water mixed solvent or deionized water to form a solution; dissolving a natural hydrophobic drug in an organic solvent to form a solution; preparing a sodium citrate-citric acid or phosphate buffer so that the pH is in the range of 7.0 to 10.0;
s2, respectively packaging the solution of the vegetable protein and the natural hydrophobic drug in the S1 into two syringes and fixing the syringes on the same syringe pump; the sodium citrate-citric acid buffer solution or the phosphate buffer solution in the step S1 is respectively packaged in two syringes and fixed on the same syringe pump;
s3, introducing the solutions in the four injectors in the S2 into a multi-channel vortex mixer through an injection pump, and taking out products at an outlet of the multi-channel vortex mixer after the interior of the mixer is stable;
s4, dialyzing and purifying the product obtained in the S3 in ultrapure water by adopting a dialysis bag with the molecular weight cutoff of 8-14 k, and obtaining the nano particles with the vegetable protein as a carrier for wrapping the natural hydrophobic medicament.
According to the invention, the concentration of the vegetable protein solution in S1 is 0.05 mg/mL-10 mg/mL.
According to the invention, the concentration of the natural hydrophobic drug in S1 is 0.5 mg/mL-3.3 mg/mL.
According to the invention, the vegetable protein is zein or soy protein isolate; the natural hydrophobic drug is curcumin, lutein or resveratrol.
According to the invention, the flow rate of the injection pump of the injector for fixing the vegetable protein and the natural hydrophobic drug is 5-30 mL/min; the flow rate of the injection pump of the injector for fixing the sodium citrate-citric acid buffer solution or the phosphate buffer solution is 10-100 mL/min.
According to the invention, in S3, the four-stream flow rate ratio of the vegetable protein, the natural hydrophobic drug and the buffer salt solution is 1 when feeding: 1:1:1 to 1:1:8:8.
in a second aspect of the present invention, there is provided a plant protein drug-loaded nanoparticle prepared by the above preparation method. Further, the particle size of the plant protein drug-loaded nano spherical particles is 30-370 nanometers.
In a third aspect of the present invention, there is provided a micromixer for preparing plant protein drug-loaded nanospheres, the micromixer comprising a multi-channel vortex mixer, a number of syringes and syringe pumps, and a number of tubes; wherein each injector communicates with an inlet end of the multi-channel vortex mixer through the conduit; four syringes, one for placing vegetable protein solution, one for placing natural hydrophobic drug solution, and two for placing buffer salt solution, two syringe pumps; wherein, two syringes for placing vegetable protein solution and natural hydrophobic drug solution are fixed on one syringe pump, and two syringes for placing buffer salt solution are fixed on the other syringe pump.
The invention has the following beneficial effects:
1. the nano particle with the vegetable protein as a carrier for wrapping the natural hydrophobic drug comprises a middle core and a shell connected outside the core, and the particle size of the nano particle refers to the whole size comprising the core and the shell. The size and drug loading of the nanoparticles can be controlled by adjusting the flow rate of the solution in the micromixer, the concentration of the drug solution, etc. The flow rate of the solution and the concentration of the solution have no absolute formula relation, and the regulation is flexible and the control is high.
2. Compared with the traditional method, the preparation method can realize the preparation of the plant protein nano particles with high drug loading, and the particle size is controllable and the particle size distribution is narrow; meanwhile, the method is favorable for continuous preparation and greatly improves the production efficiency.
Drawings
FIG. 1 is a schematic diagram of a micromixer used in the present invention.
FIG. 2 shows the result of light scattering measurement of the size of the nano-particles of the natural hydrophobic drug entrapped in the vegetable protein prepared in the exemplary embodiment by using a dynamic light scattering method.
Fig. 3 shows the stability test results of example 9 and example 10.
Detailed Description
The technical scheme of the invention is clearly and completely described in the following by specific embodiments with reference to the accompanying drawings. It is to be understood that the described embodiments are only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
FIG. 1 is a schematic diagram of a micromixer for preparing drug-loaded vegetable protein nanoparticles according to the present invention. As shown, the microreactor comprises a multi-channel vortex mixer 10, a plurality of injectors 20 and syringe pumps (not shown), and a plurality of tubes 30; wherein each injector 20 communicates with an inlet end of the multi-channel vortex mixer 10 via a conduit 30; four syringes 20, one for placing vegetable protein solution, one for placing natural hydrophobic drug solution, and two for placing buffer salt solution, and two syringe pumps; wherein two syringes 20 for placing a vegetable protein solution and a natural hydrophobic drug solution are fixed to one syringe pump, and two syringes 20 for placing a buffer salt solution are fixed to the other syringe pump.
The multichannel vortex mixer (Multi-inlet vortex mixer, MIVM mixer) used in the present invention is known in the art and is commercially available.
The experimental materials used in the following examples, such as vegetable proteins, natural hydrophobic drugs, etc., are conventionally commercially available products unless otherwise specified.
Example 1
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.1g of curcumin was dissolved in 50mL of 95% ethanol to obtain 2mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 48mL/min. Four materials adopt 1:1:4:4, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by adopting a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is measured to be 40 nanometers, the particle size distribution is 0.07, and the drug loading rate is 63 percent.
Example 2
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.15g of curcumin was taken and dissolved in 50mL of 95% ethanol to obtain a curcumin solution of 3mg/mL. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 48mL/min. Four materials adopt 1:1:4:4, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by using a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is measured to be 50 nanometers, the particle size distribution is 0.09, and the drug loading rate is 74 percent.
Example 3
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.1g of curcumin was dissolved in 50mL of 95% ethanol to obtain 2mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 72mL/min. Four materials adopt 1:1:6:6 flow rate ratio is fed, the channel is rinsed by materials, then the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after dialysis and purification are carried out in ultrapure water by adopting a dialysis bag with molecular weight cut-off of 8000-14000, the average particle size of zein nano particles is 100 nanometers, the particle size distribution is 0.10, and the drug loading rate is 60 percent.
Example 4
0.05g zein was dissolved in 50mL acetone to give a 1mg/mL zein solution. 0.15g of curcumin was taken and dissolved in 150mL of 95% ethanol to obtain 1mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, the syringe pump was set to a flow rate of 12mL/min. Four materials adopt 1:1:1:1, washing a channel with materials, feeding, collecting products at an outlet after the interior of a mixer is stable, dialyzing and purifying in ultrapure water by using a dialysis bag with a molecular weight cutoff of 8000-14000, and measuring the average particle size of zein nano particles to be 370 nanometers, the particle size distribution to be 0.06 and the drug loading to be 45 percent.
Example 5
0.05g zein was dissolved in 50mL acetone to give a 1mg/mL zein solution. 0.15g of curcumin was taken and dissolved in 150mL of 95% ethanol to obtain 1mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, the syringe pump was set to a flow rate of 12mL/min. Four materials adopt 1:1:8:8 flow rate ratio is fed, the channel is rinsed by materials, then the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after dialysis and purification are carried out in ultrapure water by adopting a dialysis bag with molecular weight cut-off of 8000-14000, the average particle size of zein nano particles is measured to be 40 nanometers, the particle size distribution is 0.09, and the drug loading rate is 45 percent.
Example 6
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.1g of curcumin was dissolved in 50mL of 95% ethanol to obtain 2mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, preparing 10mM sodium citrate-150 mu M citric acid buffer solution, and adding sodium hydroxide into the buffer solution to ensure that the final pH value of the buffer solution is in the range of 9.5-10. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 48mL/min. Four materials adopt 1:1:4:4, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by adopting a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is measured to be 30 nanometers, the particle size distribution is 0.08, and the drug loading rate is 60 percent.
Example 7
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.1g of resveratrol is taken and dissolved in 50mL of absolute ethyl alcohol to obtain 2mg/mL of resveratrol solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the resveratrol solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 60mL/min. Four materials adopt 1:1:5:5, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by adopting a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is 50 nanometers, the particle size distribution is 0.09, and the drug loading rate is 50 percent.
Example 8
0.05g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 1mg/mL zein solution. 0.1g of resveratrol is taken and dissolved in 50mL of absolute ethyl alcohol to obtain 2mg/mL of resveratrol solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the resveratrol solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 12mL/min. The sodium citrate-citric acid buffer was dispensed into two 100mL syringes and fixed on the same syringe pump, with a syringe pump flow rate of 36mL/min. Four materials adopt 1:1:3:3, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by using a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is measured to be 70 nanometers, the particle size distribution is 0.08, and the drug loading rate is up to 45 percent.
Example 9
0.024g of isolated soy protein was dissolved in 30mL of deionized water to give a 0.8mg/mL isolated soy protein solution. 0.036g of lutein was taken and dissolved in 30mL of tetrahydrofuran to give 1.2mg/mL lutein solution. An appropriate amount of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride were weighed, and PBS (disodium hydrogen phosphate 8mM, sodium dihydrogen phosphate 2mM, sodium chloride 137mM, pH 7.4) was prepared. The soybean protein isolate solution and the lutein solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, the flow rate of the syringe pump is set to be 12mL/min, PBS buffer solution is packaged in two 100mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 36mL/min. Four materials adopt 1:1:3:3, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stabilized, the products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by a dialysis bag with molecular cut-off molecular weight of 8000-14000, the average particle size of the soybean isolated protein nano particles is 100 nanometers, the particle size distribution is 0.27, and the drug loading rate is 20 percent.
Example 10
0.024g of isolated soy protein was dissolved in 30mL of deionized water to give a 0.8mg/mL isolated soy protein solution. 0.036g of lutein was taken and dissolved in 30mL of tetrahydrofuran to give 1.2mg/mL lutein solution. An appropriate amount of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride were weighed, and PBS (disodium hydrogen phosphate 8mM, sodium dihydrogen phosphate 2mM, sodium chloride 137mM, pH 7.4) was prepared. The soybean protein isolate solution and the lutein solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, the flow rate of the syringe pump is set to be 12mL/min, PBS buffer solution is packaged in two 100mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 60mL/min. Four materials adopt 1:1:5:5, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stabilized, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by using a dialysis bag with molecular cut-off molecular weight of 8000-14000, the average particle size of the soybean isolated protein nano particles is 80 nanometers, the particle size distribution is 0.23, and the drug loading rate is 20 percent.
Example 11
0.0025g zein was dissolved in 50mL 80% (V/V) ethanol water to give 0.05mg/mL zein solution. 0.025g of curcumin was dissolved in 50mL of 95% ethanol to obtain 0.5mg/mL curcumin solution. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 5mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, the syringe pump was set to a flow rate of 10mL/min. Four materials adopt 1:1:2:2, after the channel is rinsed by materials, the materials are fed, after the interior of the mixer is stable, products are collected at an outlet, and after the products are dialyzed and purified in ultrapure water by using a dialysis bag with the molecular weight cutoff of 8000-14000, the average particle size of zein nano particles is 200 nanometers, the particle size distribution is 0.139, and the drug loading rate is 80 percent.
Example 12
0.25g zein was dissolved in 50mL of 80% (V/V) ethanol aqueous solution to give 5mg/mL zein solution. 0.165g of curcumin was taken and dissolved in 50mL of 95% ethanol to obtain a curcumin solution of 3.3mg/mL. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 30mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, with the syringe pump set at a flow rate of 100mL/min. Four materials adopt 1:1:3.3:3.3, feeding after a channel is rinsed by materials, collecting products at an outlet after the interior of a mixer is stable, dialyzing and purifying the products in ultrapure water by adopting a dialysis bag with the molecular weight cut-off of 8000-14000, and measuring that the average particle size of zein nano particles is 70 nanometers, the particle size distribution is 0.105, and the drug loading rate is 38 percent.
Example 13
0.5g of zein was dissolved in 50mL of an 80% (V/V) aqueous ethanol solution to give a 10mg/mL zein solution. 0.165g of curcumin was taken and dissolved in 50mL of 95% ethanol to obtain a curcumin solution of 3.3mg/mL. Weighing a proper amount of sodium citrate and citric acid, and preparing 10mM sodium citrate-150 mu M citric acid buffer solution so that the final pH value of the buffer solution is in the range of 7.2-7.5. The zein solution and the curcumin solution are respectively packaged in two 50mL syringes and fixed on the same syringe pump, and the flow rate of the syringe pump is set to be 30mL/min. The sodium citrate-citric acid buffer solution was dispensed into two 100mL syringes and fixed on the same syringe pump, with the syringe pump set at a flow rate of 100mL/min. Four materials adopt 1:1:3.3:3.3, feeding after a channel is rinsed by materials, collecting products at an outlet after the interior of a mixer is stable, dialyzing and purifying the products in ultrapure water by adopting a dialysis bag with the molecular weight cut-off of 8000-14000, and measuring the average particle size of zein nano particles to be 80 nanometers, the particle size distribution to be 0.055 and the drug loading to be 23 percent.
Example 14
The vegetable protein nanoparticles prepared in 6 examples (examples 1,3,4,5,6 and 7) were collected and their sizes and distributions were measured by dynamic light scattering method, and the results are shown in fig. 2.
As can be seen from the results of FIG. 2, the vegetable protein nanoparticles prepared by the method have the particle size of 30-370nm, controllable particle size and narrow particle size distribution.
Example 15
The vegetable protein nanoparticles obtained in example 9 and example 10 were randomly extracted and subjected to a stability test by the following method: the prepared nanoparticle solution was subjected to a test of primary particle size and particle size distribution every 3 days.
Fig. 3 shows the stability test results of the nanoparticles of example 9 and example 10. As can be seen from the results of FIG. 3, the nano-spherical particles prepared by the method have good stability, and the particle size of the particles does not change significantly within half a month.
Claims (5)
1. The preparation method of the drug-loaded plant protein nanoparticle is characterized by comprising the following steps of:
s1, dissolving vegetable protein in an organic solvent-water mixed solvent or deionized water to form a solution; dissolving a natural hydrophobic drug in an organic solvent to form a solution; preparing a sodium citrate-citric acid or phosphate buffer so that the pH is in the range of 7.0 to 10.0;
s2, respectively packaging the solution of the vegetable protein and the natural hydrophobic drug in the S1 into two syringes and fixing the syringes on the same syringe pump; the sodium citrate-citric acid buffer solution or the phosphate buffer solution in the step S1 is respectively packaged in two syringes and fixed on the same syringe pump;
s3, introducing the solutions in the four injectors in the S2 into a multi-channel vortex mixer through an injection pump, and taking out products at an outlet of the multi-channel vortex mixer after the interior of the mixer is stable;
s4, dialyzing and purifying the product received in the S3 in ultrapure water by adopting a dialysis bag with the molecular weight cutoff of 8-14 k to obtain the nano particles of the natural hydrophobic medicament wrapped by the vegetable protein serving as a carrier;
wherein the concentration of the vegetable protein solution in the S1 is 0.05 mg/mL-10 mg/mL, and the concentration of the natural hydrophobic drug is 0.5 mg/mL-3.3 mg/mL;
s2, the flow rate of the injection pump of the injector for fixing the plant protein and the natural hydrophobic drug is 5-30 mL/min; the flow rate of the injection pump of the injector for fixing the sodium citrate-citric acid buffer solution or the phosphate buffer solution is 10-100 mL/min;
in S3, the four-stream flow rate ratio of the vegetable protein, the natural hydrophobic drug and the buffer salt solution is 1:1:1:1 to 1:1:8:8.
2. the method of claim 1, wherein the plant protein is zein or soy protein isolate; the natural hydrophobic drug is curcumin, lutein or resveratrol.
3. The drug-loaded vegetable protein nanoparticle produced by the production method according to claim 1 or 2.
4. The drug-loaded vegetable protein nanoparticle of claim 3, wherein the particle size of the drug-loaded vegetable protein nanoparticle is 30-370 nanometers.
5. A micromixer for preparing the drug-loaded vegetable protein nanoparticles of claim 3 or 4, wherein the micromixer comprises a multi-channel vortex mixer, a number of injectors and syringe pumps, and a number of pipes; wherein each injector communicates with an inlet end of the multi-channel vortex mixer through the conduit; four syringes, one for placing vegetable protein solution, one for placing natural hydrophobic drug solution, and two for placing buffer salt solution, two syringe pumps; wherein, two syringes for placing vegetable protein solution and natural hydrophobic drug solution are fixed on one syringe pump, and two syringes for placing buffer salt solution are fixed on the other syringe pump.
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| CN111317135A (en) * | 2020-02-17 | 2020-06-23 | 天津科技大学 | Method for embedding slow-release curcumin by polyphenol-modified zein nanoparticles |
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| CN111317135A (en) * | 2020-02-17 | 2020-06-23 | 天津科技大学 | Method for embedding slow-release curcumin by polyphenol-modified zein nanoparticles |
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