CN108159403B - Preparation method of polypeptide-insulin nanoparticles - Google Patents
Preparation method of polypeptide-insulin nanoparticles Download PDFInfo
- Publication number
- CN108159403B CN108159403B CN201810042408.3A CN201810042408A CN108159403B CN 108159403 B CN108159403 B CN 108159403B CN 201810042408 A CN201810042408 A CN 201810042408A CN 108159403 B CN108159403 B CN 108159403B
- Authority
- CN
- China
- Prior art keywords
- insulin
- polypeptide
- solution
- pentapeptide
- stirring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
Abstract
The invention discloses a preparation method of polypeptide-insulin nano-particles, which is characterized in that insulin monomers are dissolved in a solution, and simultaneously, a certain proportion of polypeptide with the function of inhibiting insulin fibrosis is added; and then stirring the solution for a period of time under a certain condition to enable the polypeptide and the insulin to fully react to obtain the pentapeptide-insulin supermolecule nano-particles. The method is simple to operate, the obtained supramolecular nanoparticles do not produce burst release, the sustained-release effect is good, and the polypeptide can inhibit the fibrosis of the insulin. The product has potential to solve the problems of hypoglycemia and the like caused by the burst release of insulin during clinical use and fibrosis generated in the use process of the insulin, and has wide application prospect in treating type I diabetes and type II diabetes.
Description
Technical Field
The invention belongs to the field of biomedicine, in particular to a preparation method of polypeptide-insulin nano particles with the function of inhibiting insulin monomer fibrosis, which has wide application prospect in the field of biomedicine for treating type I and type II diabetes mellitus and the like.
Background
In the course of insulin therapy, since daily insulin injection is required, causing pain and inconvenience to patients, in order to overcome these problems during the therapy, various routes of insulin administration have been developed: insulin analogs, insulin controlled release systems, gel controlled release systems, and insulin pumps. However, these products need to overcome the problem of protein aggregation in clinical applications, since protein aggregation is prone to cause certain toxic and immunogenic and other side effects. Therefore, the drug delivery system which can control the release and has a certain effect of inhibiting protein aggregation has wide development prospect and application value. In order to solve the problems, the applicant of the present invention provides a method for preparing polypeptide-insulin nanoparticles having the function of inhibiting insulin monomer fibrosis.
Disclosure of Invention
aiming at the problems in the prior art, the invention aims to provide a preparation method of polypeptide-insulin nanoparticles, the preparation method is simple to operate, the obtained supramolecular nanoparticles do not produce burst release and have good slow-release effect, and the polypeptide can inhibit the fibrosis of insulin.
Therefore, the invention adopts the following technical scheme: a preparation method of polypeptide-insulin nano-particles is characterized in that,
the method comprises the following steps: dissolving insulin monomer in the solution, and simultaneously adding polypeptide with the function of inhibiting the fibrosis of the insulin monomer in a certain proportion; insulin is a protein consisting of an A chain containing 21 amino acids and a B chain containing 30 amino acids, the insulin has the forms of monomers, dimers, hexamers and the like, in the process of forming dimers by the monomers, the B chain ends (B23-B30) of two insulin monomers form an antiparallel beta-folded structure together, and the structure is also a key step of the fibrosis formation of the insulin, when the insulin monomers are combined with an insulin receptor, the ends of the insulin B chain rotate and unfold like a hinge structure to be combined with the ends of alpha-CT of the insulin receptor, wherein the related polypeptides in the alpha-CT are phenylalanine (F), valine (V), proline (P), arginine (R) and lysine (K) (FVPPK), so that the FVRK is selected as the experimental polypeptide;
step two: and stirring the solution for a period of time under a certain condition to enable the polypeptide and the insulin monomer to fully react to obtain the pentapeptide-insulin supermolecule nano-particles.
In addition to the above technical solutions, the present invention also includes the following technical features.
In the first step, the pH value of the solution is 7.0-7.4.
Preferably, in the first step, the solution is a phosphate buffer solution containing sodium chloride, and the pH value is 7.4.
In the first step, the concentration of the insulin in the solution is 2-10 mu mol/L; the polypeptide is a short peptide of phenylalanine (F), valine (V), proline (P), arginine (R) and lysine (K) (FVPRK) which is intercepted from insulin receptor, and the concentration of the polypeptide is between 10 mu mol/L and 50 mu mol/L.
In the second step, the reaction conditions include that the temperature of the solution is 25-40 ℃.
in the second step, the stirring is constant-speed stirring or variable-speed stirring, and the stirring speed is 50-500 rpm.
in the second step, the stirring time is 1-25 h.
The invention can achieve the following beneficial effects: the polypeptide can form 100-300nm nanoparticles when added according to a certain proportion, simultaneously can inhibit insulin fibrosis, and the formed pentapeptide-insulin supramolecular nanoparticles can effectively and slowly release insulin, can be used for treating type I and type II diabetes mellitus, can slowly release and has low cytotoxicity.
Drawings
Fig. 1 is a schematic diagram of the preparation of pentapeptide-insulin supramolecular nanoparticles according to the present invention.
FIG. 2 is a graph showing fluorescence intensity changes of insulin monomer, commercial insulin, insulin monomer and polypeptide FVPRK with culture time in examples 1-3 by ThT-stained fluorescence spectroscopy.
Fig. 3 is a graph of the results of particle size measurements of the insulin fibers, pentapeptide-insulin supramolecular nanoparticles of examples 4-5.
FIG. 4 is a transmission electron microscope observation result of insulin fibers, pentapeptide-insulin supramolecular particles of examples 6 to 7.
Fig. 5 is a graph of simulated release of the commercial insulin and pentapeptide-insulin supramolecular nanoparticles of examples 8-9 in vitro.
FIG. 6 is a cytotoxicity plot of the polypeptide FVPRK of example 10.
Fig. 7 is a cytotoxicity plot of the pentapeptide-insulin supramolecular nanoparticles of example 10.
Detailed Description
The following detailed description of the embodiments of the invention is made with reference to the schematic drawings shown in fig. 1-7.
The specific preparation method comprises the following steps:
insulin monomer, polypeptide FVPRK, PBS buffer solution with pH 7.4, 100ml round-bottomed flask, magnetic stirrer, and constant temperature magnetic stirrer were prepared.
weighing 12.5mg of insulin monomer, putting the insulin monomer into a flask with a stirrer, adding 50ML PBS buffer solution with pH of 7.4, putting the mixture into a constant-temperature magnetic stirrer with adjusted temperature (37 ℃), stirring at the rotating speed of 200r/min, and sampling and testing at 0h, 1h, 2h, 3h, 4h, 5h and 6h to 25 h.
The specific preparation method comprises the following step two:
A commercial insulin, the polypeptide FVPRK, a PBS buffer solution with a pH of 7.4, a 100ml round-bottomed flask, a magnetic stirrer, and a constant-temperature magnetic stirrer were prepared
Weighing 12.5mg of insulin, putting the insulin into a flask with a stirrer, adding 50ML PBS buffer solution with the pH value of 7.4, putting the insulin into a constant-temperature magnetic stirrer with the regulated temperature (37 ℃), stirring at the rotating speed of 200r/min, and sampling and testing at 0h, 1h, 2h, 3h, 4h, 5h and 6h to 25 h.
The third concrete preparation method comprises the following steps:
Preparing insulin monomer, polypeptide FVPRK, PBS buffer solution with pH of 7.4, 100ml round-bottom flask, magnetic stirrer and constant-temperature magnetic stirrer
Weighing 12.5mg of insulin monomer and 6.9mg of polypeptide FVPRK, putting the insulin monomer and the polypeptide FVPRK into a flask with a stirrer, adding 50ML of PBS buffer solution with the pH value of 7.4, putting the mixture into a constant-temperature magnetic stirrer with the regulated temperature (37 ℃), stirring at the rotating speed of 200r/min, and sampling and testing at 0h, 1h, 2h, 3h, 4h, 5h and 6h to 25 h.
Example four of the specific preparation method:
A commercial insulin, the polypeptide FVPRK, a PBS buffer solution with a pH of 7.4, a 100ml round-bottomed flask, a magnetic stirrer, and a constant-temperature magnetic stirrer were prepared
Weighing 12.5mg of insulin, putting the insulin into a flask with a stirrer, adding 50ML PBS buffer solution with pH of 7.4, putting the solution into a constant-temperature magnetic stirrer with adjusted temperature (37 ℃), stirring at the rotating speed of 200r/min, stopping reaction when 15 hours later, and sampling and testing.
The concrete preparation method comprises the following step five:
Preparing insulin monomer, polypeptide FVPRK, PBS buffer solution with pH of 7.4, 100ml round-bottom flask, magnetic stirrer and constant-temperature magnetic stirrer
Weighing 12.5mg of insulin monomer and 6.9mg of polypeptide FVPRK, putting the insulin monomer and the polypeptide FVPRK into a flask with a stirrer, adding 50ML of PBS buffer solution with the pH value of 7.4, putting the mixture into a constant-temperature magnetic stirrer with the regulated temperature (37 ℃), stirring at the rotating speed of 200r/min, stopping reaction when 15 hours later, and sampling and testing.
The concrete preparation method comprises the following step six:
A commercial insulin, the polypeptide FVPRK, a PBS buffer solution with a pH of 7.4, a 100ml round-bottomed flask, a magnetic stirrer, and a constant-temperature magnetic stirrer were prepared
Weighing commercial insulin 12.5mg into a flask with a stirring bar, adding 50ML PBS buffer solution with pH of 7.4, placing into a constant temperature magnetic stirrer with adjusted temperature (37 ℃), stirring at the speed of 200r/min, stopping reaction at 15h, and sampling for testing.
the specific preparation method comprises the following seventh step:
Preparing insulin, polypeptide FVPRK, PBS buffer solution with pH of 7.4, 100ml round-bottom flask, magnetic stirrer, and constant-temperature magnetic stirrer
Weighing 12.5mg of insulin monomer and 6.9mg of polypeptide FVPRK, putting the insulin monomer and the polypeptide FVPRK into a flask with a stirrer, adding 50ML of PBS buffer solution with the pH value of 7.4, putting the mixture into a constant-temperature magnetic stirrer with the regulated temperature (37 ℃), stirring at the rotating speed of 200r/min, stopping reaction when 15 hours later, and sampling and testing.
The concrete preparation method comprises the following eight steps:
Weighing 3mg of pentapeptide-insulin supramolecular nanoparticle supramolecular nanoparticles, dissolving in 1ml of 5mM PBS buffer, loading in 1.5ml of pp tube to release the internal liquid, releasing the external liquid to 50ml of 10mM PBS buffer containing 0.01% w/v sodium azide at pH 7.4 and release temperature of 37 ℃, and oscillating. 1ml of the release sheath was removed at intervals of response time and then 3ml of fresh PBS buffer was added dropwise with a pipette. Samples were taken and tested quantitatively for insulin concentration by HPLC.
Example nine of the specific preparation method:
Insulin 3mg was weighed, dissolved in 1ml of 5mM PBS buffer and the release inner solution was filled in 1.5ml pp tubes, the release outer solution was 50ml of 10mM PBS buffer containing 0.01% w/v sodium azide at pH 7.4 and release temperature 37 ℃ and shaken. 1ml of the release sheath was removed at intervals of response time and then 3ml of fresh PBS buffer was added dropwise with a pipette. Samples were taken and tested quantitatively for insulin concentration by HPLC.
Example of the specific preparation method tenth:
According to the method of the present invention, pentapeptide-insulin supramolecular nanoparticles, polypeptide FVPRK, PBS buffer solution with pH 7.4, 96-well plate were prepared.
Weighing 0.1mg of polypeptide FVPRK and pentapeptide-insulin supramolecular nanoparticles, dissolving a sample by using a culture medium, performing membrane filtration sterilization, respectively adding the sample into a 96-well plate, and testing the cytotoxicity by using cck-8.
the foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (1)
1. A preparation method of polypeptide-insulin nanoparticles is characterized by comprising the following steps:
The method comprises the following steps: dissolving insulin monomer in the solution, and simultaneously adding pentapeptide with the function of inhibiting the fibrosis of the insulin monomer; the pH value of the solution is 7.0-7.4, the solution is a phosphate buffer solution containing sodium chloride, the pH value is 7.4, the concentration of insulin in the solution is 2-10 mu mol/L, wherein the pentapeptide is a section of pentapeptide FVPRK intercepted from an insulin receptor, and the concentration is 10 mu mol/L-50 mu mol/L;
Step two: stirring the solution for 1-25h to allow the pentapeptide and the insulin monomer to fully react to obtain the pentapeptide-insulin supermolecule nano-particles; the reaction conditions include that the temperature of the solution is 25-40 ℃, the stirring is constant-speed stirring or variable-speed stirring, and the stirring speed is 50-500 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810042408.3A CN108159403B (en) | 2018-01-17 | 2018-01-17 | Preparation method of polypeptide-insulin nanoparticles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810042408.3A CN108159403B (en) | 2018-01-17 | 2018-01-17 | Preparation method of polypeptide-insulin nanoparticles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108159403A CN108159403A (en) | 2018-06-15 |
| CN108159403B true CN108159403B (en) | 2019-12-10 |
Family
ID=62515069
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810042408.3A Active CN108159403B (en) | 2018-01-17 | 2018-01-17 | Preparation method of polypeptide-insulin nanoparticles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108159403B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102293748A (en) * | 2011-07-25 | 2011-12-28 | 华南理工大学 | Oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof |
| CN103169946A (en) * | 2011-12-20 | 2013-06-26 | 武汉光谷世傲生物科技有限公司 | Application of Safenour cyclopeptide in oral insulin medicine for treating diabetes |
-
2018
- 2018-01-17 CN CN201810042408.3A patent/CN108159403B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102293748A (en) * | 2011-07-25 | 2011-12-28 | 华南理工大学 | Oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof |
| CN103169946A (en) * | 2011-12-20 | 2013-06-26 | 武汉光谷世傲生物科技有限公司 | Application of Safenour cyclopeptide in oral insulin medicine for treating diabetes |
Non-Patent Citations (1)
| Title |
|---|
| Inhibition of Insulin Amyloid Fibrillation by a Novel Amphipathic Heptapeptide;Bhisma N. Ratha等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20161104;第291卷(第45期);23545-23556、补充材料 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108159403A (en) | 2018-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | A fluorinated ionic liquid-based activatable 19F MRI platform detects biological targets | |
| CN110613844B (en) | Mini combined adjuvant nanoparticle and preparation method and application thereof | |
| CN110183601A (en) | Preparation method and application of covalent organic framework material containing acylhydrazone bonds and disulfide bonds | |
| CN105859990B (en) | The polymer of side chain sulfur-bearing caprylyl, its preparation method and polymer vesicle prepared therefrom and its application | |
| CN108159403B (en) | Preparation method of polypeptide-insulin nanoparticles | |
| US10550205B2 (en) | Process for preparing water-dispersible single-chain polymeric nanoparticles | |
| CN110452390A (en) | A kind of insulin intelligent drug delivery preparation | |
| CN106880846B (en) | Tumor-targeted multifunctional nano drug delivery system, preparation method and application | |
| CN108888773B (en) | Self-assembled spherical medicine nano preparation and preparation method and application thereof | |
| CN109223827A (en) | Application of the water-soluble fullerene structure in the drug of preparation treatment pulmonary fibrosis | |
| CN108187060B (en) | Drug carrier, drug preparation and preparation method | |
| US20240000897A1 (en) | Injectable biodegradable polymeric complex for glucose-responsive insulin delivery | |
| CN106176579B (en) | A kind of preparation with pH response characteristic injectable Nanometer composite hydrogel system | |
| CN110974784B (en) | Multifunctional polymer micelle for combined delivery of chemotherapeutic drugs and gene editing system, and preparation method and application thereof | |
| CN113754795A (en) | Sulfonated hyaluronic acid compound, preparation method and application thereof | |
| CN102614523B (en) | A kind of dexamethasone macromolecule prodrug and preparation method thereof | |
| CN112190565B (en) | Kidney-targeted nano particle capable of reducing toxicity of gefitinib and preparation method thereof | |
| CN109875978A (en) | A kind of preparation method of the polyacrylic acid can be used for medicament slow release-tocopherol succinate self assembly medicine-carried system | |
| CN108721605A (en) | A kind of nano-particle being mounted with insulin and its application | |
| CN109568592B (en) | Nano gel NG1 and application thereof | |
| US20210052749A1 (en) | Amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions | |
| CN103720663B (en) | A kind of follicle stimulating hormone sustained-release micro-spheres and preparation method thereof | |
| CN107540740B (en) | Compound, preparation method and MRI (magnetic resonance imaging) application thereof | |
| CN105561330B (en) | A kind of human calcitonin-Cucurbituril compound formulation and preparation method thereof | |
| CN103957890B (en) | A kind of preparation method and application of Needleless injection injection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |