CN111920784A - Intelligent nanorod for oral insulin and preparation method thereof - Google Patents
Intelligent nanorod for oral insulin and preparation method thereof Download PDFInfo
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- 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
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- 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
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
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- 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
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- 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
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5115—Inorganic compounds
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- 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
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses an oral insulin intelligent nanorod and a preparation method thereof, and relates to the technical field of biological medicines. The intelligent nanorod consists of an inner core layer, a middle layer and an outer shell layer; wherein the inner core layer is formed by mixing insulin and a micromolecule permeation enhancer, the middle layer is a mesoporous silica rod-shaped material, and the outer shell layer is a pH-sensitive enteric coating material; the oral insulin intelligent nanorod has pH sensitivity, the encapsulation rate can reach more than 95%, the drug loading rate can reach 15%, the mesoporous silica and the insulin can show strong charge repulsion force at the pH value of more than 6, the release of the insulin in intestinal juice can be promoted, meanwhile, the permeation enhancer can not only open the opening of the intestinal epithelial cell in tight connection, but also can cause reversible disturbance to cell membranes, so that the medicine can be better absorbed, the bioavailability of the oral medicine is improved, the process is simple, the conditions are mild, the operation is easy, and the oral administration of the insulin is expected to be realized.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a method for encapsulating insulin, and particularly relates to a preparation method of an oral insulin intelligent nanorod.
Background
Diabetes has become an important disease which endangers human health, and the most effective treatment means at present is subcutaneous insulin injection, but the administration mode is very inconvenient, the administration is required for multiple times every day, and meanwhile, the long-term injection brings great pain to patients, and complications such as hypoglycemia reaction, subcutaneous fat atrophy, hypertrophic malnutrition and the like occur, so that the compliance of the patients is weakened. Compared with the injection administration route, the oral administration route is more convenient and has higher patient compliance, but in the oral administration process, due to the existence of a large amount of proteolytic enzyme in the gastrointestinal tract, insulin can be hydrolyzed into small molecular polypeptide or amino acid to lose activity, and the drug effect cannot be exerted.
Currently, oral insulin dosage forms need to overcome the absorption barrier of polypeptide protein drugs existing in the intestinal mucosal epithelium, and also need to prevent insulin from being decomposed and digested by various proteases in the digestive tract. Although the targeted therapeutic effect can be achieved, the oral insulin liposome which is researched intensively at present is not easy to be absorbed by oral administration. Although the oral insulin dosage forms are various, the oral insulin dosage forms are not actually applied to clinic, mainly because the bioavailability of the insulin is low, the preparation process is complex and takes long time, and the product stability is poor.
Therefore, the development of an oral insulin intelligent nanorod with high oral utilization rate and simple and convenient method operation and a preparation method thereof become problems which need to be solved by the technical personnel in the field.
Disclosure of Invention
In view of the above, the present invention provides an oral insulin intelligent nanorod, which aims to solve the problems in the prior art.
In order to achieve the above purpose, the invention provides the following technical scheme:
an oral insulin intelligent nanorod is composed of an inner core layer, an intermediate layer and an outer shell layer; wherein the inner nuclear layer is prepared from insulin and a small molecule permeation enhancer according to a mass ratio of 1: (1-10), wherein the middle layer is a mesoporous silica rod-shaped material, and the outer shell layer is a pH-sensitive enteric coating material.
Preferably, the isoelectric point range of the insulin is 4-7, the insulin is natural insulin and/or modified insulin, and the small molecule permeation enhancer is alkyl acid containing carboxyl and a short carbon chain.
Preferably, the surface potential of the mesoporous silica rod-shaped material is-60 mV to-20 mV, the pore diameter is 3-8 nm, the length is 50-800 nm, and the length-diameter ratio is 2-15.
More preferably, the length of the mesoporous silica rod-shaped material is 100-500 nm, and the length-diameter ratio is 2-5.
Preferably, the enteric coating material mainly comprises polyacrylic acid and/or hydroxypropyl methylcellulose, the enteric coating material is stable in a pH range of 1.0-4.0 and is dissolved in a pH range of 5.5-7.0, and the molecular weight of the enteric coating material is 10000-100000.
Preferably, the molecular weight of the insulin is 5000-10000; the number of carboxylic acid groups in the alkyl acid of the micromolecular permeation enhancer is 1-3, and the number of carbon atoms in a short carbon chain of the micromolecular permeation enhancer is 5-10.
It should be noted that the development of inorganic nanomaterials provides a new approach to oral insulin, in which mesoporous silica carriers have unique physicochemical properties: the pore diameter of 2-50 nm is large enough to load protein drug molecules in the drug molecules, small enough to avoid the protein drug from being degraded by enzyme in the gastrointestinal tract, and can limit the structure transformation or aggregation of the protein drug, so that the dispersibility of the protein drug can be improved, and the stability of the protein drug can be improved; the mesoporous silica has low toxicity and good biocompatibility, can be absorbed by gastrointestinal tracts after being taken orally and enter the systemic circulation, is mainly distributed in the liver after 24 hours, and is finally excreted by urine and excrement. The pathological experiment proves that the mesoporous silicon dioxide has good histocompatibility, and the cytotoxicity experiment also shows that the mesoporous silicon dioxide has no obvious toxicity to Caco-2, HT-29, HepG2 and other cells.
However, the mesoporous silica can only realize large drug loading, and release can not be controlled, so that the mesoporous silica is coated with an enteric coating material hydroxypropyl methylcellulose phthalate (HP55) which has the advantages of insolubility in an acidic solution and easy dissolution in an alkaline solution, can protect insulin from the influence of acidic pH in simulated gastric juice (SGF, pH value of 1.2), and can successfully release drugs in simulated intestinal juice (SIF, pH value of 7.4).
The invention combines the advantage that the mesoporous silicon dioxide carrier is beneficial to protecting the stability of protein and the coating characteristic of hydroxypropyl methylcellulose phthalate to control the release of oral insulin, and a mesoporous silica carrier with large aperture (8nm) is designed according to the space size (6.23nm multiplied by 6.18nm multiplied by 4.47nm) of the insulin, and the surface of the solution is coated with hydroxypropyl methyl cellulose phthalate material to finally prepare a pH sensitive type silicon dioxide insulin-loaded system, the drug loading of the system reaches 15 percent, the encapsulation rate can reach 95 percent, and because the synthesized SBA-15 has uniform aperture and 8nm aperture, the system can sufficiently contain insulin, meanwhile, the permeation enhancer is encapsulated to promote the absorption of the medicine, and the coating method is simple and low in cost, and has a profound application prospect in realizing the batch production of the oral insulin.
The invention also provides a preparation method of the oral insulin intelligent nanorod, and the preparation method is simple in process, mild in condition, easy to operate and suitable for industrial popularization and application.
A preparation method of an oral insulin intelligent nanorod comprises the following steps:
(1) adding insulin, a small molecule permeation enhancer and mesoporous silica into a hydrochloric acid solution, and magnetically stirring and mixing to obtain a primary dispersion liquid;
(2) performing ultrasonic cavitation and dispersion on the primary dispersion liquid, centrifuging and washing, and dispersing in a PBS (phosphate buffer solution) solution to obtain a secondary dispersion liquid;
(3) dissolving the enteric coating material in a mixed solvent of dichloromethane/acetone to obtain an enteric coating material solution;
(4) mixing the second-stage dispersion liquid with the enteric coating material solution, pouring the mixture into a macromolecular surfactant solution, stirring and dispersing, and removing a mixed solvent in a rotary evaporation mode to obtain a third-stage dispersion liquid;
(5) and freeze-drying the third-level dispersion liquid to finally obtain the oral insulin intelligent nanorod.
Preferably, in the step (1), the mass ratio of insulin to the small molecule permeation enhancer is 1: (1-10), wherein the mass ratio of the insulin to the mesoporous silica is 0.125-1; and the pH value of the hydrochloric acid solution is 5-6, and the magnetic stirring and mixing time is 3-12 h.
In addition, when the concentration of the insulin is 0.24mg/mL, the concentration of the micromolecule permeation enhancer (alkyl acid) is 2.4mg/mL, and the mass ratio of the insulin to the mesoporous silica nano-rods is 0.75: 1, when the pH value of the hydrochloric acid solution is 5.0, the drug loading rate of the mesoporous silica nano-rod on insulin is the highest; and the magnetic stirring mixing time is preferably 4h, and the adsorption process of the mesoporous silica nanorods to the insulin simultaneously has a desorption process, so that the mesoporous silica nanorods reach equilibrium within 4 h.
Preferably, in the step (2), the ultrasonic time is 1-15 min, the ultrasonic power is 100-800W, and the pH range of the PBS solution is 7.2-7.4.
It should be noted that when the power of ultrasonic cavitation is 500W, the ultrasonic time is 10min, the centrifugal speed is 6500r/min, and the time is 5min, the solid-liquid separation is thorough, and the reduction of the yield caused by too much throwing-off of insulin can be effectively avoided.
Preferably, in the step (3), the concentration of the enteric coating material is 1-2 mg/mL, and the volume ratio of dichloromethane to acetone in the mixed solvent is (0.3-1): 1.
It should be noted that when the volume ratio of dichloromethane to acetone is 3:1 and the concentration of the enteric coating material is 5mg/mL, the coating effect of the enteric coating material is better.
Preferably, in the step (4), the mass ratio of the secondary dispersion liquid to the enteric coating material is 1 (1-3), the stirring speed is 100-800 r/min, and the stirring time is 10-60 min; the molecular weight range of the macromolecular surfactant is 8000-30000, and the concentration of the macromolecular surfactant solution is 0.1-1%.
In addition, the enteric coating material HP55 is a pH sensitive material which is insoluble in acid and soluble in a medium with a pH value of more than 5.0; experiments prove that the enteric coating material HP55 is added in a proper amount, the slow release effect is not good when the amount is too small, and the slow release is realized when the amount is too large.
Specifically, the technical principle of the preparation method of the intelligent oral insulin nanorod is as follows:
firstly synthesizing a silicon nanorod with the aperture exceeding 7nm in an acid environment, encapsulating a certain amount of insulin and alkyl acid in the nanorod by a physical impregnation method, and then encapsulating hydroxypropyl methyl cellulose phthalate on the surface of the silicon nanorod by a multiple emulsion solvent volatilization method to obtain the oral insulin nanorod.
The isoelectric point of the silicon nanorods is 2.5, and the isoelectric point of the insulin is 5.5, so that the silicon nanorods and the insulin have a large electrostatic repulsion effect in an intestinal fluid environment (pH is 5.2-7.5) and can promote the release of the drug, and the electrostatic repulsion force between the insulin and the mesoporous silica is small in a gastric fluid environment (pH is 1.0-2.5); in addition, the hydroxypropyl methyl cellulose phthalate coated on the surface of the nanorod has pH sensitivity, and the enteric coating material keeps stable in the pH range of 1.0-4.0 and dissolves in the pH range of 5.5-7.0, so that insulin can be better protected and released in an intestinal juice environment to form a multi-stage pH response release system finally.
Meanwhile, as the silicon nanorods are used as an anionic carrier to enhance mucus penetrability, and small molecule penetrating agents such as alkyl acid are added into the carrier, the silicon nanorods can open the opening of epithelial cell tight connection to cause reversible interference on cell membranes, so that the drug can penetrate through the epithelial cells of small intestines as much as possible and enter capillaries to improve the bioavailability of the oral drug.
Compared with the prior art, the invention discloses and protects an oral insulin intelligent nanorod and a preparation method thereof, and has the advantages that:
1. the method comprises the steps of synthesizing mesoporous silica under an acidic condition by a sol-gel method, then encapsulating insulin and a permeation enhancer by ultrasonic and magnetic stirring, finally coating hydroxypropyl methyl cellulose phthalate on the surface of the mesoporous silica by a double emulsion solvent volatilization method, and freeze-drying to obtain the oral insulin intelligent nanorod.
2. The prepared intelligent oral insulin nanorod has high oral bioavailability, good biocompatibility, excellent performance and controllable appearance; the drug loading capacity can be improved by changing the crystallization temperature (60-130 ℃) to obtain larger pore diameter, and the invention adopts the enteric polymer as the pore blocking material, so that the gastric juice environment is closed, and the intestinal juice environment is opened, thereby realizing intelligent control.
3. The intelligent oral insulin nanorod prepared by the invention has the drug loading rate of 15.1% and the encapsulation rate of 97.9%, and can realize effective embedding of insulin;
by adopting the mesoporous silica as a carrier material, the mesoporous silica has good biocompatibility, so that the mesoporous silica is applied to the loading of hydrophobic drugs, the high drug loading capacity of the drugs is realized, the water solubility and the dissolution rate are enhanced, the drug release and the loading are facilitated, and the insulin can be effectively protected;
the hydroxypropyl methyl cellulose phthalate coated on the surface can effectively control the release of insulin in simulated gastric juice, and specifically comprises the following steps: only 10% of insulin is released within 12h, 43.5% of insulin is released within 1h in gastric juice, and the release amount can reach 60.1% after 12h of continuous release;
the enteric coating material HP55 avoids the damage of gastric acid to insulin and the degradation of pepsin to insulin to a certain extent, and improves the bioavailability.
4. The preparation method disclosed and protected by the invention is simple, convenient and reliable, mild in condition and short in experimental period, and is expected to realize market application in batch production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a Fourier infrared spectrum of various materials of the feedstock of example 1 of the present invention; wherein, fig. 1(a) is a fourier infrared spectrum of the insulin-loaded mesoporous silica/hydroxypropylmethylcellulose phthalate composite of example 1, fig. 1(B) is a fourier infrared spectrum of hydroxypropylmethylcellulose phthalate (HP55), and fig. 1(C) is a fourier infrared spectrum of mesoporous silica.
FIG. 2 is an SEM photograph of mesoporous silica and oral insulin nanorods according to example 1 of the present invention; wherein FIG. 2(A) is an SEM image of mesoporous silica, and FIG. 2(B) is an SEM image of oral insulin nanorods.
FIG. 3 is an electrogram of mesoporous silica and insulin in media solutions of different pH in accordance with example 1 of the present invention.
Fig. 4 is a controlled release profile of oral insulin smart nanorods in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) in example 1 of the present invention.
Fig. 5 is a controlled release profile of oral insulin smart nanorods coated with hydroxypropylmethylcellulose phthalate (20mg) in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) of example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it should be understood 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 obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention discloses a rapeseed oil flavor component extraction method combining coupling probe type ultrasonic auxiliary extraction and solvent extraction evaporation, which has the characteristics of non-thermal effect, low energy consumption, high efficiency, real extracted substance and the like, can obviously shorten the pretreatment time of a sample, and can identify more flavor components under the same qualitative condition compared with solid phase micro-extraction.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.
Example 1
1. Preparation of mesoporous silica nano-rod loaded with insulin and alkyl acid
Dissolving 10mg of insulin and 10mg of alkyl acid in hydrochloric acid solution with pH of 2, wherein the mass ratio of insulin to alkyl acid is 1:1, the concentration of insulin is 2.4mg/mL, and completely dissolving the insulin by magnetic stirring at 25 ℃ to form insulin solution; weighing 20mg of mesoporous silica, adding the mesoporous silica into hydrochloric acid solution with pH being 5, adding 10mL of insulin solution, adjusting the pH to 5 by using sodium hydroxide, magnetically stirring the solution at 25 ℃ for 4 hours, centrifuging the solution at 6500r/min for 5 minutes, washing the precipitate for 3 times by using a certain amount of PBS solution, and freeze-drying the precipitate to obtain the uncoated drug-loaded nanorod.
2. Preparation of hydroxypropyl methyl cellulose phthalate coated drug-loaded nanorod
Weighing 20mg of drug-loaded nanorod, dispersing in 5mL of hydrochloric acid solution with pH being 5, taking the solution as an internal water phase, weighing 5mg of hydroxypropyl methylcellulose phthalate, adding into 4mL of dichloromethane/acetone mixed solution to serve as an oil phase, adding the internal water phase into the solution after the polymer is completely dissolved, quickly and uniformly stirring to form multiple emulsion, preparing 1% (w/v) of PVA solution as an external water phase, quickly pouring the multiple emulsion into 10mL of PVA solution, magnetically stirring for 1h at room temperature, removing dichloromethane/acetone by rotary evaporation at 30 ℃, and finally freeze-drying to obtain the oral insulin intelligent nanorod.
3. Determination of drug loading rate and encapsulation efficiency of insulin-loaded nanorods
The drug loading percentage and the mass encapsulation efficiency are commonly used for expressing the drug loading capacity of the nanorods, and the mass encapsulation efficiency and the drug loading percentage are calculated by measuring free insulin in supernate after washing the insulin-loaded mesoporous silica by using a high performance liquid chromatography through an indirect method. And measuring the content of insulin in the supernatant, and further calculating the drug loading percentage and the mass encapsulation efficiency. Wherein, the drug loading percentage refers to the percentage of the drug quantity wrapped into the nano-rod to the total mass (carrier + wrapped drug), the mass encapsulation rate refers to the mass percentage of the drug quantity wrapped into the nano-rod and the drug dosage, and the calculation formula of the mass encapsulation rate is as follows:
wherein M isGeneral assemblyIs the initial insulin mass (mg), VSupernatant fluidVolume of supernatant (mL), CSupernatant fluidThe concentration of insulin in the supernatant (mg/mL), WNano-rodThe mass (mg) of the obtained nanorods.
The drug loading mass percentage of the insulin-loaded nano rod is measured to be 15.3 percent, and the mass encapsulation rate is measured to be 95.34 percent.
And, the above-mentioned pancreatic isletsFig. 1 shows fourier infrared spectra of mesoporous silica/hydroxypropylmethylcellulose phthalate complex of cellulose, hydroxypropylmethylcellulose phthalate (HP55), and mesoporous silica. As can be seen from FIG. 1, by comparison with FIG. 1(B), the compound to which HP55 was added was at 3359 cm-1，2934cm-1，1735cm-1All have reinforcement and compare it with figure 1(C), at 1089cm-1The peak at (a) was clearly reduced due to coating with HP 55.
In addition, fig. 2 is an SEM image of mesoporous silica and oral insulin smart nanorods of example 1; the mesoporous silica surface contour shown in fig. 2(a) is clear and is a nano rod, while the oral insulin intelligent nanorod shown in fig. 2(B) has a blurred surface, but the basic contour rod is not changed, and the surface interface blur of the nanorod is caused by coating the mesoporous silica surface with hydroxypropyl methyl cellulose phthalate (HP 55).
And, FIG. 3 is a potential diagram of mesoporous silica and insulin in medium solutions of different pH in example 1 of the present invention. As shown in figure 3, the isoelectric point of mesoporous silica (SBA-15) is 3-4, the isoelectric point of insulin is 5-6, the drug loading rate is maximum when the pH of the medium solution is the isoelectric point of insulin, and the drug loading effect is best, because the insulin is uncharged under the condition and is easier to load, the mesoporous silica (SBA-15) has the highest drug loading rate and the best drug loading effect
4. Insulin-loaded nanorod simulated release
A hydrochloride buffer solution with a pH value of 1.2 and a phosphate buffer solution with a pH value of 7.4 are respectively used as an in vitro simulated gastric fluid and a simulated intestinal fluid, and insulin release behaviors in micelles are analyzed, which are specifically described as follows:
accurately weighed 3.0mg of insulin-loaded nanorods were dissolved in 3mL of simulated gastric fluid (0.15MHCl, 0.05MKCl, pH1.2) and simulated intestinal fluid (8g NaCl, 0.2g KCl, 1.44g Na), respectively2HPO4，0.24gKH2PO4pH7.4), releasing at 37 deg.C, horizontally shaking for 100 times/min, taking out sample liquid at different time points of 1h, 2h, 4h, 6h, 8h and 10h, centrifuging, collecting supernatant, determining insulin concentration, and making into oral liquidThe cumulative percent release of insulin over different times was calculated by the formula:
wherein V is the volume of a release medium (simulated gastric fluid or simulated intestinal fluid, mL), C is the concentration (mg/mL) of insulin in supernatant obtained by centrifuging the released sample liquid, W is the mass (mg) of the insulin-loaded enteric-coated microspheres, and LC is the drug-loaded mass percentage (%) of the insulin-loaded nanorods.
The release result shows that the insulin-loaded nanorod prepared by the invention is slowly released in simulated gastric fluid, only 10% of insulin is released within 12 hours, 43.5% of insulin is released within 1 hour in intestinal fluid, and the release amount can reach 61.2% after continuous release for 12 hours.
Wherein, fig. 4 is a controlled release profile of the oral insulin smart nanorods in example 1 in the release profiles of simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). And as can be seen from fig. 4, the mass of the oral insulin intelligent nanorod in simulated gastric fluid released within 2h reaches 36%, the accumulated release mass of insulin released within 2h reaches 56% in simulated intestinal fluid, and the repulsive force in gastric fluid detected by potential is not large in intestinal fluid, so that the electrostatic action plays a driving role in the release process, and from the release curve, the release of the mesoporous silica loaded with drug has a burst release effect, and the maximum release effect is reached at 2 h.
Fig. 5 is a controlled release profile of oral insulin smart nanorods coated with hydroxypropylmethylcellulose phthalate (20mg) in example 1 in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). As can be seen from figure 5, the mass of the intelligent oral insulin nanorod is less than 10% in simulated gastric fluid within 12h, the hydroxypropyl methylcellulose phthalate is rapidly dissolved due to the alkaline pH in the simulated intestinal fluid, the accumulated release mass of the insulin released within 2h reaches 42%, and as can be seen from figure 5, the oral insulin has an obvious controlled release effect after being coated with the hydroxypropyl methylcellulose phthalate, and the release amount in the intestinal fluid is about 5 times that of the gastric fluid.
Example 2
The procedure is as in example 1, 10mg of insulin and 25mg of alkyl acid are dissolved in hydrochloric acid solution with pH 2, the ratio of insulin/alkyl acid is 1:2.5 (mass ratio), and the concentration of insulin is 1.2mg/mL at 25 ℃, and the solution is completely dissolved by magnetic stirring to form insulin solution; weighing 20mg of mesoporous silica nanorods, adding the mesoporous silica nanorods into hydrochloric acid solution with pH of 5, adding 10mL of insulin solution, adjusting the pH to 5 by using sodium hydroxide, magnetically stirring the mesoporous silica nanorods at 25 ℃ for 4h, centrifuging the mesoporous silica nanorods at 6500r/min for 5 minutes, washing the mesoporous silica nanorods for 3 times, precipitating the mesoporous silica nanorods by using a certain amount of PBS solution, and freeze-drying the mesoporous silica nanorods to obtain drug-loaded nanorods, wherein the drug-loaded nanorods are 14.68% and the encapsulation rate is 81.54%.
Example 3
The procedure is as in example 1, 10mg of insulin and 50mg of alkyl acid are dissolved in hydrochloric acid solution with pH 2, the ratio of insulin/alkyl acid is 1:5 (mass ratio), and the concentration of insulin is 4.8mg/mL at 25 ℃, and the solution is completely dissolved by magnetic stirring to form insulin solution; weighing 20mg of mesoporous silica nanorods, adding the mesoporous silica nanorods into hydrochloric acid solution with pH of 5, adding 10mL of insulin solution, adjusting the pH to 5 by using sodium hydroxide, magnetically stirring for 4h at 25 ℃, centrifuging for 5min at 6500r/min, washing for 3 times by using a certain amount of PBS solution, precipitating, and freeze-drying to obtain the drug-loaded nanorods, wherein the drug-loaded nanorods are 11.31% and the encapsulation rate is 95.55%.
Example 4
The procedure is as in example 1, 10mg of insulin and 100mg of alkyl acid are dissolved in hydrochloric acid solution with pH 2, the ratio of insulin/alkyl acid is 1:10 (mass ratio), and the concentration of insulin is 9.6mg/mL at 25 ℃, and the solution is completely dissolved by magnetic stirring to form insulin solution; weighing 20mg of mesoporous silica nanorods, adding the mesoporous silica nanorods into hydrochloric acid solution with pH of 5, adding 10mL of insulin solution, adjusting the pH to 5 by using sodium hydroxide, magnetically stirring for 4h at 25 ℃, centrifuging for 5min at 6500r/min, washing for 3 times by using a certain amount of PBS solution for precipitation, and freeze-drying to obtain the drug-loaded nanorods, wherein the drug-loaded nanorods are measured to have the drug-loaded rate of 10.96% and the encapsulation rate of 97.32%.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. An oral insulin intelligent nanorod is characterized by consisting of an inner core layer, a middle layer and an outer shell layer; wherein the inner nuclear layer is prepared from insulin and a small molecule permeation enhancer according to a mass ratio of 1: (1-10), wherein the middle layer is a mesoporous silica rod-shaped material, and the outer shell layer is a pH-sensitive enteric coating material.
2. The oral insulin intelligent nanorod according to claim 1, wherein the isoelectric point of insulin is 4-7, the insulin is natural insulin and/or modified insulin, and the small molecule permeation enhancer is an alkyl acid containing carboxyl and a short carbon chain.
3. The intelligent nanorod for oral insulin according to claim 1, wherein the surface potential of the mesoporous silica rod-like material is-60 mV to-20 mV, the pore diameter is 3nm to 8nm, the length is 50nm to 800nm, and the length-diameter ratio is 2 nm to 15.
4. The intelligent nanorod according to claim 1, wherein the enteric coating material is mainly composed of polyacrylic acids and/or hydroxypropyl methylcellulose, is stable at a pH of 1.0-4.0, and is soluble at a pH of 5.5-7.0, and has a molecular weight of 10000-100000.
5. The oral insulin intelligent nanorod according to claim 1, wherein the molecular weight of the insulin is 5000-10000; the number of carboxylic acid groups in alkyl acid of the micromolecule permeation enhancer is 1-3, and the number of carbon atoms in a short carbon chain of the micromolecule permeation enhancer is 5-10.
6. The method for preparing the oral insulin intelligent nanorod according to claim 1, comprising the following steps:
(1) adding insulin, a small molecule permeation enhancer and mesoporous silica into a hydrochloric acid solution, and magnetically stirring and mixing to obtain a primary dispersion liquid;
(2) performing ultrasonic cavitation and dispersion on the primary dispersion liquid, centrifuging and washing, and dispersing in a PBS (phosphate buffer solution) solution to obtain a secondary dispersion liquid;
(3) dissolving an enteric coating material in a mixed solvent of dichloromethane/acetone to obtain an enteric coating material solution;
(4) mixing the second-stage dispersion liquid with the enteric coating material solution, pouring the mixture into a macromolecular surfactant solution, stirring and dispersing, and removing a mixed solvent in a rotary evaporation mode to obtain a third-stage dispersion liquid;
(5) and freeze-drying the third-level dispersion liquid to finally obtain the oral insulin intelligent nanorod.
7. The method for preparing intelligent nanorods of insulin for oral administration according to claim 6, wherein in the step (1), the mass ratio of insulin to small molecule permeation enhancer is 1: (1-10), wherein the mass ratio of the insulin to the mesoporous silica is 0.125-1; and the pH value of the hydrochloric acid solution is 5-6, and the magnetic stirring and mixing time is 3-12 h.
8. The method for preparing intelligent nanorods of insulin for oral administration according to claim 6, wherein in the step (3), the concentration of the enteric coating material is 1-2 mg/mL, and the volume ratio of dichloromethane and acetone in the mixed solvent is (0.3-1): 1.
9. The method for preparing intelligent nanorods of insulin for oral administration according to claim 6, wherein in the step (4), the mass ratio of the secondary dispersion liquid to the enteric coating material is 1 (1-3).
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