CN115569188A - Preparation method and application of insulin-controllable nano particles - Google Patents

Abstract

The invention discloses a preparation method and application of Nanoparticles (NPs) capable of controllably releasing insulin. Dissolving protamine in water to prepare a protamine solution, adding an insulin solution consisting of insulin, water and HCl, stirring and mixing by a vortex mixer, dropwise adding the mixture into a Fucoidin (FU) solution, and mixing and stirring to obtain the nanoparticles. The nanoparticles are used for carrying insulin, so that the insulin can be prevented from being enzymolyzed by gastrointestinal protease, the stability of the insulin in the gastrointestinal tract is improved, the insulin can be favorably absorbed and operated by the intestinal tract, and therapeutic medicaments can enter systemic circulation from the gastrointestinal tract.

Description

Preparation method and application of insulin-controllable nano particles

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method and application of nanoparticles capable of controlling insulin release.

Background

Diabetes is a chronic disease that seriously threatens the health of residents and is also an independent risk factor for causing various diseases. The existing administration mode of insulin is subcutaneous injection, but long-term injection can cause tissue edema, hard injection part, malnutrition, tolerance and other adverse consequences, and brings pain and inconvenience to diabetics. Oral administration is a non-invasive and convenient administration mode, improves the compliance of patients, and is one of the first-choice administration routes of protein drugs. The route of oral insulin administration mimics the physiological mechanism of endogenous insulin secretion by the liver after absorption by the gastrointestinal tract and is expected to protect pancreatic cells from autoimmune destruction. However, oral insulin administration often exhibits very low bioavailability due to its relatively large molecular mass, poor absorption by intestinal epithelial cells, and rapid degradation by enzymes and gastric acids in the gastrointestinal tract. Research shows that embedding insulin with the carrier can avoid degradation of insulin by gastrointestinal proteinase, raise the stability of insulin in gastrointestinal tract, prolong insulin acting time and promote the therapeutic medicine to enter body circulation from gastrointestinal tract.

The drug carrier produced by the nanotechnology is an innovative drug product, the structure of the nano particles is stable, insulin can be protected to a certain extent, and the insulin is not digested and degraded, and meanwhile, the nano particles have the advantages of drug targeting, high-efficiency packaging, improvement of curative effect, avoidance of toxicity and the like. Patent CN102908332B discloses an enteric coated capsule containing cationic nanoparticles for oral insulin delivery encapsulating a plurality of cationic nanoparticles, wherein the enteric coated capsule comprises a pH-sensitive coating layer to rapidly dissolve and continuously release the cationic nanoparticles in the upper segment of the small intestine, wherein the pH-sensitive coating layer is selected from polymers consisting of: hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (HPMCAS), and acrylic resin. In this patent, the release of insulin from the particles is slow in acidic media and fast in neutral media. However, the preparation method of the particles is complex, and the preparation cost is relatively high.

The marine polysaccharide has abundant sources and good biocompatibility, biodegradability and bioactivity, and has attracted great interest in the development of nano-drug carriers. Among them, fucoidan (FU) has attracted much attention because of its various biological activities such as antioxidant, anti-inflammatory, anticoagulant, antiviral, and antitumor. FU is derived from marine brown algae, mainly comprises L-fucose and sulfate groups, has good water solubility, and can promote the preparation of a nano-composite between FU and other positively charged molecules due to the negative charge of the FU caused by a large amount of sulfate groups. FU has the effects of regulating and maintaining blood glucose level, and can prevent diabetes-related complications by inhibiting the activities of amylase and cohesive glycosidase, and stimulate insulin secretion to protect pancreas.

Protamine has strong positive charge, so that the carried medicine can pass through cell membrane, and compared with non-target medicine, the nano particle carrier based on FU has stronger penetrating power in vivo and in vitro, so that the FU/protamine nano particle has better medicine transfer characteristic compared with the traditional FU/chitosan nano particle.

Disclosure of Invention

The invention aims to provide a nanoparticle capable of controlling insulin release, which has good water solubility, can effectively prevent insulin from being enzymolyzed by gastrointestinal protease, improves the stability of the insulin in gastrointestinal tracts and has better drug delivery characteristics.

In order to achieve the purpose, the invention adopts the technical scheme that: a nanoparticle capable of controlling insulin release is prepared by using protamine and fucoidin as composite carrier of insulin.

Specifically, the Zeta potential absolute value of the nano particles in an acidic environment and a neutral environment is more than 15mV.

Specifically, the nanoparticles are complete in structure and regular spherical in an acidic environment, and are cracked in a neutral environment, so that the shape is incomplete.

The preparation method of the nanoparticle capable of controlling the release of the insulin provided by the invention comprises the following steps:

(1) Respectively preparing a protamine solution, a fucoidin solution and an insulin solution;

(2) Adding the protamine solution into the insulin solution, and stirring and mixing by adopting a vortex mixer;

(3) And (3) dropwise adding the mixture obtained in the step (2) into the fucoidin solution, and stirring and mixing the mixture by adopting a vortex mixer to obtain the nano particles.

Specifically, the protamine is dissolved in deionized water to prepare a protamine solution with the protamine mass concentration of 1.5 g/L; dissolving fucoidin in deionized water to prepare fucoidin solution with the mass concentration of 4.5 g/L; dissolving insulin in HCl, and then adding deionized water to prepare an insulin solution with the insulin mass concentration of 3g/L, wherein the molar concentration of the hydrochloric acid is 0.01mol/L; the volume of the protamine solution and the volume of the insulin solution in the step (2) are 1:1, the stirring and mixing time is 2min, the volume of the mixture and the volume of the fucoidin in the step (3) are 2:1, and the stirring and mixing time is 3min.

The invention further claims the application of the nano particles in the construction of a drug oral delivery system, in particular for the controlled release of insulin.

By implementing the technical scheme of the invention, the following beneficial effects can be achieved:

(1) The characterization analysis can observe that the nanoparticles have stable structures in an acid environment, are not or not easily degraded by gastric acid, and can well protect insulin from entering systemic circulation through gastric juice.

(2) The observation of electron microscope pictures in different environments shows that the nanoparticles have complete structures in gastric juice and break in intestinal juice, so that the purpose of controlling the nanoparticles to release insulin in intestinal tracts is achieved.

Drawings

FIG. 1 is Zeta potential diagram of nanoparticles under different pH conditions.

FIG. 2 is a Fourier transform infrared spectrum of nanoparticles under different pH conditions.

FIG. 3 is a transmission electron micrograph of nanoparticles.

FIG. 4 is a transmission electron micrograph of the nanoparticles in the gastric juice prosthesis at 2 h.

FIG. 5 is a transmission electron micrograph of the nanoparticles in the artificial intestinal juice for 12 h.

FIG. 6 is an insulin standard curve.

FIG. 7 is the concentration of insulin released by nanoparticles and pure insulin in SGF.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example prepared a nanoparticle with controlled release of insulin.

15mg of protamine was weighed and dissolved in 10mL of deionized water to prepare a protamine solution. Fucoidan solution was prepared by dissolving 45mg of fucoidan in 10mL of deionized water. An insulin solution was prepared by dissolving 30mg of insulin in 1mL of 0.01mol/L HCl and then adding 9mL of deionized water. Adding 1mL of protamine solution into 1mL of insulin solution, stirring and mixing for 2min by using a vortex mixer, dropwise adding the mixture into 1mL of fucoidin solution, and stirring for 3min by using the vortex mixer to obtain the nano particles.

Example 2

This example measured the particle size and Zeta potential of the nanoparticles. The preparation of the nanoparticles used is described in example 1.

Storing the nanoparticles in a 37 ℃ incubator at pH1.2, pH6.8 and pH7.4, transferring 1mL of sample solution into a cuvette with an optical path of 1cm by using a liquid transfer gun at 0h, 1h, 3h, 6h and 12h, measuring the particle size and the Zeta potential of the nanoparticles by using a nanoparticle size potentiometer at a measurement temperature of 25 ℃, repeatedly measuring each sample for three times, and averaging. The results of the particle size measurement of the present invention are shown in table 1.

Table 1: particle size of nanoparticles at different pH and time

The residence time of the drug in the stomach is typically 1-2 hours, so that only 3 hours are detected at pH 1.2. The smaller the particle size of the nanoparticles, the larger the free surface, the stronger the adsorption, and the more stable the nanoparticles. As can be seen from the above table, the nanoparticles are substantially stable in gastric fluid at pH1.2, and then stable again with significant changes in both intestinal fluid at pH6.8 and body fluid at pH 7.4.

The Zeta potential test results of the present invention are shown in fig. 1. The nanoparticles with Zeta potential absolute values of more than 15mV are relatively stable, and it can be seen from FIG. 1 that the nanoparticles are relatively stable at pH1.2, pH6.8, pH7.4, do not coagulate or agglomerate, and are more stable at pH 7.4.

Example 3

This example performed FTIR spectroscopy on nanoparticles. The preparation of the nanoparticles used is described in example 1.

Adjusting pH of the nanoparticle solution to pH1.2, pH6.8, and pH7.4, respectively, lyophilizing, and mixing 3mg of the nanoparticles with 150mg of dry pure KBrPressing into transparent sheet with tablet press, and measuring by Fourier infrared spectrometer at 400cm ^-1 ～4000cm ^-1 FTIR spectroscopy was performed. The results of FTIR spectroscopy are shown in FIG. 2.

As can be seen from FIG. 2, the nanoparticles are very stable at pH1.2, relatively stable at pH6.8, and unstable at pH 7.4. It can be concluded that the nanoparticles are very stable in gastric fluid, relatively stable in intestinal fluid and unstable in body fluids.

Example 4

This example observes the microscopic morphological structure of nanoparticles. See example 1 for the preparation of the nanoparticles used.

Respectively storing the nanoparticle solution in artificial gastric juice for 2h and artificial intestinal juice for 12h in 37 deg.C, and performing morphological analysis on the micro-and nano-scale microstructures of the nanoparticles with Transmission Electron Microscope (TEM). For TEM imaging, TEM analysis samples were prepared by immersing copper grids in NPs suspension, dried at room temperature, and prepared using 300 mesh agar lace carbon film copper grids. The TEM images are shown in fig. 3, 4, and 5.

As can be seen from fig. 3, 4 and 5, the nanoparticles have complete shape and intact structure after being stored in the artificial gastric juice for 2 hours, and are in the shape of regular spheres and can be completely stored; the nano particles are cracked after being stored in the artificial intestinal juice for 12 hours, and the shape is incomplete and cannot be completely stored.

Example 5

This example measured the in vitro release effect of nanoparticles at different pH. The preparation of the nanoparticles used is described in example 1.

2mL of nanoparticles were placed in a dialysis bag of 3000kDa, and 3mL of artificial gastric juice (SGF, pH 2.0) was added to the outside of the dialysis bag and incubated at 37 ℃. Taking the liquid outside the dialysis bag at 0h, 0.5h, 1h, 1.5h and 2h respectively, centrifuging at 8000rpm for 20min by using a high-speed refrigerated centrifuge, collecting 1mL of supernatant, and supplementing with equivalent volume of artificial gastric juice. Adding 200 μ L sodium carbonate solution into the supernatant, filtering with membrane, placing into a sample bottle, and detecting with high performance liquid chromatograph. 2mL of nanoparticles were placed in a 3000kDa dialysis bag, and 3mL of artificial intestinal fluid (SIF, pH 6.8) was added to the dialysis bag and stored at 37 ℃. 1mL of supernatant was collected at 0h, 1h, 3h, 6h, and 12h, respectively. Adding 200 μ L sodium carbonate solution into the supernatant, filtering with membrane, placing into a sample bottle, and detecting with high performance liquid chromatograph. And then the nano particles are replaced by pure insulin solution, and the steps are repeated. The insulin standard curve is shown in figure 6. The equation y =653.51x-54221 for calculating the insulin concentration can be obtained from the insulin standard curve of fig. 6, and the insulin concentrations released by the nanoparticles and the pure insulin in SGF and SIF are calculated as shown in fig. 7, table 2 and table 3.

Table 2: concentration of insulin released by nanoparticles and pure insulin in SGF

Pure insulin is not present in artificial intestinal fluids because insulin is completely digested in the intestine by digestive fluids. As can be seen from table 2 and fig. 7, the nanoparticles released less insulin than the pure insulin solution in the artificial gastric juice. It can be seen from table 3 that insulin concentration was detected in the artificial intestinal juice. Therefore, the nano-particles have a certain protection effect on insulin, and the insulin can smoothly reach the intestinal tract by wrapping the insulin, so that the decomposition of gastric acid and enzyme on the insulin is reduced.

Table 3: concentration of insulin released by nanoparticles and pure insulin in SIF

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A nanoparticle capable of controlling insulin release is characterized in that the nanoparticle is prepared by taking protamine and fucoidin as a composite carrier of insulin.

2. Nanoparticles according to claim 1, characterized in that the Zeta potential absolute value of the nanoparticles in acidic and neutral environments is greater than 15mV.

3. Nanoparticles according to claim 1, wherein the nanoparticles are structurally sound in a regular spherical shape in an acidic environment; cracking occurs in a neutral environment and the morphology is incomplete.

4. Nanoparticles according to claim 2 or 3, characterised in that the acidic environment has a pH of 1.2 and the neutral environment has a pH of 6.8 or 7.4.

5. A method for preparing nanoparticles capable of controlling and releasing insulin is characterized by comprising the following steps:

(1) Respectively preparing a protamine solution, a fucoidin solution and an insulin solution;

(2) Adding the protamine solution into the insulin solution, and stirring and mixing;

(3) And (3) dropwise adding the mixture obtained in the step (2) into the fucoidin solution, and stirring and mixing to obtain the nano particles.

6. The method according to claim 5, wherein the step (1) is to dissolve protamine in deionized water to prepare a protamine solution with a protamine mass concentration of 1.5 g/L; dissolving fucoidin in deionized water to prepare fucoidin solution with the mass concentration of fucoidin of 4.5 g/L; dissolving insulin in HCl, and adding deionized water to prepare an insulin solution with the insulin mass concentration of 3g/L, wherein the molar concentration of the hydrochloric acid is 0.01mol/L.

7. The method according to claim 5, wherein the volume of the protamine solution and the volume of the insulin solution in the step (2) are 1:1, and the stirring and mixing time is 2min.

8. The method according to claim 5, wherein the volume of the mixture in the step (3) and the volume of the fucoidan are 2:1, and the mixing time is 3min under stirring.

9. Use of the nanoparticle of claim 1 for the construction of an oral drug delivery system.

10. The use according to claim 9, wherein the nanoparticles are used for the controlled release of insulin.

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