CN116271095A - Intestinal adhesive enzymolysis-resistant gel microcapsule oral delivery system for bioactive macromolecular medicament - Google Patents
Intestinal adhesive enzymolysis-resistant gel microcapsule oral delivery system for bioactive macromolecular medicament Download PDFInfo
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- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6925—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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Abstract
The invention relates to an intestinal adhesion enzymolysis-resistant gel microcapsule oral delivery system of bioactive macromolecular drugs, which comprises the following components: the chitosan is adsorbed on the surface of the sodium alginate drug-loaded gel microcapsule and grafted with digestive enzyme inhibitor; the sodium alginate medicine-carrying gel microcapsule is composed of a sodium alginate wall material and bioactive macromolecular medicines embedded in the sodium alginate wall material. The oral delivery system has effects of resisting gastric acid and digestive enzyme degradation, and has intestinal adhesion, and can maintain protease activity in digestive tract for a long time. The invention also relates to a preparation method of the intestinal adhesion enzymolysis-resistant gel microcapsule oral delivery system of the bioactive macromolecular drug, and the preparation method does not introduce organic solvents in the preparation process, and has low material biotoxicity and safe and reliable product.
Description
Technical Field
The invention belongs to the technical field of pharmacy, and relates to a gel microcapsule oral delivery system of a bioactive macromolecular medicament with an anti-proteolysis effect and intestinal adhesion and a preparation method thereof.
Background
Biological macromolecular drugs have become important components of innovative drugs and play an increasingly important role in the treatment of some key diseases. Meanwhile, the biological macromolecule medicine has huge molecular weight and fragile structure, and faces to complex gastrointestinal tract environment and various absorption barriers, and the biological macromolecule is easily damaged in the gastrointestinal tract. Therefore, the bioavailability of the biomacromolecule drug through an oral mode is extremely low, and the 'holy cup' in the field of high-efficiency oral delivery of protein drugs and drug delivery is realized. Among the reasons for reducing the oral effects of biological macromolecules, one important reason is the degradation of digestive enzymes. The intestinal tract has various digestive enzymes, and can be combined with specific sites of bioactive macromolecular drugs with high efficiency to destroy the structure of the bioactive macromolecular drugs, so that the bioactive macromolecular drugs lose activity. Therefore, how to resist degradation by digestive enzymes in the digestive tract is a long felt but unsolved problem.
Disclosure of Invention
One of the objects of the present invention is to provide an enteric adhesive enzymolysis resistant gel microcapsule oral delivery system for bioactive macromolecular drugs, which aims at solving the problems existing in the prior art. The oral delivery system has effects of resisting gastric acid and digestive enzyme degradation, and has intestinal adhesion, and can maintain activity of bioactive macromolecular drug in digestive tract for a long time.
The second object of the invention is to provide a preparation method of an intestinal adhesion enzymolysis-resistant gel microcapsule oral delivery system of bioactive macromolecular drugs, wherein no organic solvent is introduced in the preparation process of the preparation method, the material biotoxicity is low, and the product is safe and reliable.
To this end, a first aspect of the present invention provides a gel microcapsule oral delivery system for a bioactive macromolecular drug, comprising:
the chitosan is adsorbed on the surface of the sodium alginate drug-loaded gel microcapsule and grafted with digestive enzyme inhibitor;
the sodium alginate medicine-carrying gel microcapsule is composed of a sodium alginate wall material and bioactive macromolecular medicines embedded in the sodium alginate wall material.
According to the invention, the sodium alginate wall material is formed by crosslinking sodium alginate solution and metal ions; preferably, the metal ions include Ca 2+ And/or Zn 2+ 。
In some embodiments of the invention, the digestive enzyme inhibitor is a substance having an activity of inhibiting human gastrointestinal proteases; preferably, the digestive enzyme inhibitor comprises one or more of aprotinin, soybean trypsin inhibitor, chymotrypsin and pepsin inhibitor.
In some embodiments of the invention, the bioactive macromolecular drug comprises a nucleic acid, a polypeptide, a protein; preferably, the bioactive macromolecular drug comprises one or more of GLP-1 and analogues thereof, insulin, glucagon, sialin-4, salmon calcitonin, interferon, various antibodies, trypsin, thrombin, lysozyme, catalase, protease inhibitors and catalase.
According to the invention, the chitosan grafted with the digestive enzyme inhibitor is formed by using a phenolic precursor as a crosslinking medium to crosslink and fix the digestive enzyme inhibitor on the chitosan under the catalysis of polyphenol oxidase.
In some embodiments of the invention, the polyphenol oxidase is an enzyme capable of oxidizing a phenol or polyphenol to form a corresponding quinone by molecular oxygen; further preferably, the polyphenol oxidase comprises monophenol monooxidase (tyrosinase), bisphenol oxidase (catechol oxidase) and laccase.
In some embodiments of the invention, the phenolic precursor is a natural substance having phenolic hydroxyl groups; preferably, the phenolic precursor comprises methyl gallate, gallic acid, chlorogenic acid, arbutin, caffeic acid, p-benzoquinone, catechol.
In a second aspect, the present invention provides a method for preparing a gel microcapsule oral delivery system of a bioactive macromolecular drug, comprising:
step L, under the catalysis of polyphenol oxidase, using a phenol precursor as a crosslinking medium to crosslink and fix a digestive enzyme inhibitor on chitosan, so as to obtain chitosan grafted with the digestive enzyme inhibitor;
step M, embedding bioactive macromolecular drugs into the sodium alginate wall material to obtain sodium alginate drug-loaded gel microcapsules;
and step N, coating chitosan grafted with a digestive enzyme inhibitor on the surface of the sodium alginate drug-loaded gel microcapsule to obtain the gel microcapsule system for orally delivering the bioactive macromolecular drug.
According to the invention, in step L, the phenol precursor and the digestive enzyme inhibitor are dissolved in chitosan solution and mixed uniformly, polyphenol oxidase is added into the mixed solution, and the mixture is incubated at room temperature, dialyzed, filtered and freeze-dried to obtain the chitosan grafted with the digestive enzyme inhibitor.
In some embodiments of the invention, the chitosan solution is formed by dissolving chitosan in 0.1M hydrochloric acid solution and adjusting ph=5.5 to 6.5 with 0.01M NaOH solution; preferably, the chitosan solution has a concentration of 0.1wt% to 2wt%.
In some embodiments of the invention, in step L, the mass ratio of phenolic precursor to chitosan is 1 (1-10).
In some embodiments of the invention, in step L, the mass ratio of digestive enzyme inhibitor to chitosan is 5:1 to 1:2.
In some embodiments of the invention, in step L, the enzyme activity unit (U) of the polyphenol oxidase: phenolic precursor mole number= (1000000 ～ 8000000): 1.
In some embodiments of the invention, in step L, the room temperature incubation time is from 6 to 24 hours.
According to the invention, in the step M, the bioactive macromolecular drug is dispersed in sodium alginate solution to form bioactive macromolecular drug-sodium alginate dispersion, and the bioactive macromolecular drug-sodium alginate dispersion is mixed and crosslinked with metal ion solution through processing and molding treatment to obtain the sodium alginate drug-carrying gel microcapsule; preferably, the method of the process molding treatment includes an emulsification method, a instillation method or a spraying method.
In some embodiments of the invention, in step M, the sodium alginate solution is formed by dissolving sodium alginate in water; preferably, the concentration of the sodium alginate solution is 0.5-5 wt%.
In some embodiments of the invention, in step M, the content of the bioactive macromolecular drug in the bioactive macromolecular drug-sodium alginate dispersion is 0.5wt% to 40wt%.
In some embodiments of the invention, in step M, the concentration of the aqueous metal ion solution is 0.5wt% to 3wt%;
in some embodiments of the invention, in step M, the volume ratio of the bioactive macromolecular drug-sodium alginate dispersion to the metal ion aqueous solution is 1 (0.1-10).
According to the invention, in the step N, the sodium alginate medicine-carrying gel microcapsule is dispersed into a solution of chitosan grafted with a digestive enzyme inhibitor, the solution is incubated at room temperature, the chitosan grafted with the digestive enzyme inhibitor is adsorbed on the surface of the sodium alginate medicine-carrying gel microcapsule through electrostatic action, and the gel microcapsule system for orally delivering the bioactive macromolecular medicine is obtained through filtration, washing and drying.
In some embodiments of the invention, in step N, the solution of the digestive enzyme inhibitor grafted chitosan is formed by dissolving the digestive enzyme inhibitor grafted chitosan in an acetic acid-sodium acetate buffer solution having a ph=4 to 6; preferably, the content of the chitosan grafted with the digestive enzyme inhibitor in the solution of the chitosan grafted with the digestive enzyme inhibitor is 0.1 to 2 weight percent; and/or the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.2M;
in some embodiments of the present invention, in the step N, the mass-to-volume ratio of the sodium alginate drug-loaded gel microcapsule to the solution of chitosan grafted with the digestive enzyme inhibitor is 1 (1-10) g/mL;
in some embodiments of the invention, in step N, the incubation time at room temperature is 5-60min.
The invention has the following advantages:
(1) The chitosan grafted with the digestive enzyme inhibitor is compounded with the sodium alginate microcapsule, so that the microcapsule has excellent effects of resisting gastric acid and digestive enzyme degradation, has intestinal adhesion, and can keep protease activity in the digestive tract for a long time;
(2) The technology crosslinks the digestive enzyme inhibitor to chitosan through an enzyme catalysis method, the enzyme catalysis crosslinking is realized in a mild water phase environment, no organic solvent is introduced, and the crosslinking process is green and pollution-free;
(3) The adopted polyphenol oxidase, phenolic precursors, sodium alginate and chitosan are natural products, the material biotoxicity is low, and the products are safe and reliable.
Drawings
The invention will be described in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic representation of the preparation of an enteric adhesive enzymolysis resistant gel microcapsule oral delivery system for bioactive macromolecular drugs.
Fig. 2 is a scanning electron microscope image of an intestinal adhesion anti-enzymolysis gel microcapsule oral delivery system for bioactive macromolecular drugs, wherein a is a sodium alginate gel drug-carrying microcapsule, B is a chitosan encapsulated sodium alginate gel drug-carrying microcapsule, and C is a chitosan encapsulated sodium alginate gel drug-carrying microcapsule grafted with aprotinin.
FIG. 3 is a graph showing the IR spectrum before and after chitosan grafted aprotinin.
Fig. 4 shows the comparison of the anti-enzymolysis effect of the chitosan-encapsulated sodium alginate gel drug-loaded microcapsule grafted with aprotinin in the invention with that of the common sodium alginate gel drug-loaded microcapsule in artificial gastric juice and artificial intestinal juice.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail below with reference to the accompanying drawings. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I terminology
The term "water" as used herein refers to one or more of deionized water, distilled water and ultrapure water under the conditions not specifically described or specified.
II. Embodiment
As described above, one of the reasons for reducing the oral effects of biomacromolecules is the degradation of digestive enzymes. The intestinal tract has various digestive enzymes, and can be combined with specific sites of bioactive macromolecular drugs with high efficiency to destroy the structure of the bioactive macromolecular drugs, so that the bioactive macromolecular drugs lose activity. Therefore, how to resist the degradation of trypsin in the digestive tract is a long felt but yet unsolved problem. In view of this, the present inventors have conducted extensive studies on an enzymolysis-resistant oral delivery system of a bioactive macromolecular drug.
Preferably, the inventors devised the coacervation as a drug carrier. The hydrogel forms a stable three-dimensional network structure through various crosslinking modes by a hydrophilic polymer chain, and plays an important role in the field of drug delivery as a common drug delivery system. Due to the rich pore channel structure and the high surface area associated with the pore channel structure, the hydrogel has extremely high loading capacity on medicines; in addition, the material has soft touch feeling and low interfacial tension in an aqueous medium, is similar to the characteristics of tissues, and has good biocompatibility due to the hydrophilic and stable material properties; at the same time, by selecting the appropriate hydrogel components, the hydrogels can be made to have a variety of environmental responsivity. In addition, a portion of the cationic polymer forms a hydrogel that binds negatively charged mucin in the intestine, thereby providing the gel with intestinal adhesion and allowing the loaded drug to function in the intestine for a longer period of time.
Chitosan and alginate are hydrogel components which are widely researched and applied, are natural polymer materials, have good biocompatibility and extremely low biotoxicity, and have the advantages of simple preparation, good safety guarantee and simple production process, so that the hydrogel has natural advantages which are more similar to the application. In addition, these two drugs have unique advantages in the field of oral macromolecular administration: sodium alginate is an anionic polymer, and the pH in the stomach is lower than the acid dissociation constant (pKa), so that the formed hydrogel can keep a stable collapse state in the stomach, and the sodium alginate chain is ionized in the intestinal tract along with the rise of the pH in the environment to be greater than the pKa, so that the sodium alginate absorbs water to swell, and the internal medicine is continuously released, so that the pH responsiveness is excellent.
Meanwhile, chitosan has unique properties as a cationic polymer, and the mucin surface in the intestinal mucus layer has negative charges, so that the chitosan with positive charges on the surface can be combined with mucin under the action of electrostatic force, and then is adhered in the intestinal tract, and is kept in the digestive tract for a longer time; meanwhile, the chitosan has the function of reversibly opening the tight connection of intestinal epithelial cells, thereby promoting the drug bypass transportation and increasing the absorption availability. There have been many studies to demonstrate that sodium alginate gel microcapsules can have good intestinal adhesion after encapsulation with chitosan, prolonging the release time of the drug in the intestinal tract.
However, the present inventors have studied and found that, although chitosan-encapsulated sodium alginate microcapsules have many advantages, their loose pore structure cannot resist degradation by digestive enzymes, and thus cannot maintain the activity of bioactive macromolecular drugs in the intestinal tract for a long period of time.
The inventors contemplate that protease inhibitors can bind reversibly or irreversibly to specific sites on proteases, thereby inactivating the enzymes and protecting the protein drug from degradation by them. Several small molecule inhibitors have been studied extensively at present, including camostat mesylate, bacitracin, soybean trypsin inhibitor, aprotinin, etc., which have remarkable effects and become an important method for protecting protein drugs from degradation by digestive enzymes.
However, currently, digestive enzyme inhibitors are not widely used in industry because, although they are well-defined, they may have a great loss due to rapid dilution, digestion and absorption of digestive fluids, thereby compromising their effectiveness. Although these factors can be overcome by using high doses of inhibitors, such high doses also present significant safety concerns, and studies have shown that the pancreas can easily compensate for the presence of inhibitors by activating feedback loops that increase protease secretion, so that the use of protease inhibitors in large amounts can lead to pancreatic hypertrophy and hyperplasia, while the use of small molecule inhibitors in large amounts, such as bacitracin, can potentially cause nephrotoxicity. In addition, since these inhibitors can prevent degradation and absorption of other proteins than drugs, thereby altering metabolism in the gastrointestinal tract, the use of digestive enzyme inhibitors in large amounts is not an excellent approach to solve this problem. Therefore, a new application mode is found for the digestive enzyme inhibitor, the damage of the dilution and absorption of the digestive tract to the effect is reduced, and the digestive enzyme inhibitor is a direction with research and development value and is also a subject with application potential.
Based on the above, the present inventors have studied and devised to combine a digestive enzyme inhibitor with an intestinal adhesion system to solve the above-mentioned problems. The inventor develops an intestinal adhesion hydrogel microcapsule through research, and skillfully grafts a digestive enzyme inhibitor on chitosan on the surface layer of the intestinal adhesion hydrogel microcapsule in an enzyme catalytic crosslinking mode, so that the intestinal adhesion hydrogel microcapsule has excellent effect of resisting intestinal digestive enzyme degradation. The present inventors have further studied and found that an orally administered microcapsule which is resistant to gastric acid, resistant to degradation by digestive enzymes and has intestinal adhesion can be prepared by immobilizing a digestive enzyme inhibitor on chitosan by a green harmless enzyme crosslinking method and adsorbing it on the surface of sodium alginate microcapsule, which can effectively deliver macromolecular drugs such as polypeptides, proteins and the like, and thus the present invention has been achieved.
Thus, the gel microcapsule oral delivery system of the bioactive macromolecular drug according to the present invention comprises: the chitosan (layer) is adsorbed on the surface of the sodium alginate medicine-carrying gel microcapsule and grafted with digestive enzyme inhibitor; the sodium alginate medicine-carrying gel microcapsule is composed of a sodium alginate wall material and bioactive macromolecular medicines embedded in the sodium alginate wall material. Therefore, the gel microcapsule oral delivery system of the bioactive macromolecular drug is also called a chitosan encapsulated sodium alginate gel drug-carrying microcapsule grafted with aprotinin or a chitosan-sodium alginate drug-carrying microcapsule grafted with aprotinin on the surface.
According to the invention, the sodium alginate wall material is formed by crosslinking sodium alginate solution with metal ions through an emulsification method, a instillation method or a spraying method.
In the present invention, the metal ion exists in the form of a metal salt, which includes Ca 2+ And/or Zn 2+ Such as calcium chloride, zinc nitrate, zinc acetate, etc.
In some embodiments of the invention, the digestive enzyme inhibitor is a substance having an activity of inhibiting human gastrointestinal proteases; preferably, the digestive enzyme inhibitor comprises one or more of aprotinin, soybean trypsin inhibitor, chymotrypsin and pepsin inhibitor.
In some embodiments of the invention, the bioactive macromolecular drug comprises a nucleic acid, a polypeptide, a protein; preferably, the bioactive macromolecular drug comprises one or more of GLP-1 and analogues thereof, insulin, glucagon, sialin-4, salmon calcitonin, interferon, various antibodies, trypsin, thrombin, lysozyme, catalase, protease inhibitors and catalase.
According to the invention, the chitosan grafted with the digestive enzyme inhibitor is formed by using a phenolic precursor as a crosslinking medium to crosslink and fix the digestive enzyme inhibitor on the chitosan under the catalysis of polyphenol oxidase.
In some embodiments of the invention, the polyphenol oxidase is an enzyme capable of oxidizing a phenol or polyphenol to form a corresponding quinone by molecular oxygen; further preferably, the polyphenol oxidase comprises monophenol monooxidase (tyrosinase), bisphenol oxidase (catechol oxidase) and laccase.
In some embodiments of the invention, the phenolic precursor is a natural substance having phenolic hydroxyl groups; preferably, the phenolic precursor comprises methyl gallate, gallic acid, chlorogenic acid, arbutin, caffeic acid, p-benzoquinone, catechol. The phenolic precursors oxidize phenolic hydroxyl groups under the catalysis of polyphenol oxidase, react with amino groups on chitosan according to the Schiff base reaction principle or the Michael addition principle, so that the phenolic precursors are connected to the chitosan, and the digestive enzyme inhibitor and the chitosan are grafted together through the combination of benzene rings in the phenolic precursors and the digestive enzyme inhibitor.
In order to realize the technical scheme of the invention, the invention provides a preparation method of a gel microcapsule oral delivery system of a bioactive macromolecular drug, as shown in figure 1, as can be seen from figure 1, the preparation method comprises the following steps:
step L, under the catalysis of polyphenol oxidase, using a phenol precursor as a crosslinking medium to crosslink and fix a digestive enzyme inhibitor on chitosan, so as to obtain chitosan grafted with the digestive enzyme inhibitor;
step M, embedding bioactive macromolecular drugs into the sodium alginate wall material to obtain sodium alginate drug-loaded gel microcapsules;
and step N, coating chitosan grafted with a digestive enzyme inhibitor on the surface of the sodium alginate drug-loaded gel microcapsule to obtain the gel microcapsule system for orally delivering the bioactive macromolecular drug.
According to the invention, in step L, the phenol precursor and the digestive enzyme inhibitor are dissolved in chitosan solution and mixed uniformly, polyphenol oxidase is added into the mixed solution, and the mixture is incubated at room temperature, dialyzed, filtered and freeze-dried to obtain the chitosan grafted with the digestive enzyme inhibitor.
Specifically, in step L, the preparation method of the chitosan grafted with the digestive enzyme inhibitor includes:
(1) A certain amount of chitosan was dissolved in a 0.1M hydrochloric acid solution, and the ph=5.5 to 6.5 of the above solution was adjusted with a 0.01M NaOH solution, and the final mass concentration was set to be in the range of 0.1wt% to 2wt%.
(2) The phenolic precursor is dissolved in the chitosan solution according to the mass ratio of the phenolic precursor to the chitosan of 1 (1-10).
(3) According to the mass ratio of the digestive enzyme inhibitor to the chitosan of 5:1-1:2, dissolving the digestive enzyme inhibitor in the chitosan solution, and uniformly mixing. Next, according to the unit of enzyme activity of polyphenol oxidase (U):
(4) According to the enzyme activity unit (U) of polyphenol oxidase, the mole number of phenolic precursor is = (1000000 ～ 8000000), 1, adding polyphenol oxidase into the above-mentioned mixed liquor, incubating for 6-24h at room temperature, dialyzing, filtering and freeze-drying so as to obtain the invented chitosan grafted with digestive enzyme inhibitor.
According to the invention, in the step M, the bioactive macromolecular drug is dispersed in sodium alginate solution to form bioactive macromolecular drug-sodium alginate dispersion, and the bioactive macromolecular drug-sodium alginate dispersion is mixed and crosslinked with metal ion solution through processing and molding treatment to obtain the sodium alginate drug-carrying gel microcapsule; preferably, the method of the process molding treatment includes an emulsification method, a instillation method or a spraying method.
Specifically, in the step M, the method for preparing the sodium alginate drug-loaded gel microcapsule comprises the following steps:
(1) A certain amount of sodium alginate is dissolved in water to prepare sodium alginate solution with the final mass concentration of 0.5-5 wt%. Dissolving or dispersing the bioactive macromolecular drug in sodium alginate solution to prepare bioactive macromolecular drug-sodium alginate dispersion liquid, wherein the content of the bioactive macromolecular drug is 0.5-40 wt%;
(2) Mixing the above-mentioned biological active macromolecular medicine-sodium alginate dispersion liquor with 0.5wt% -3 wt% of Ca by means of emulsification method, instillation method or spray method 2+ Or Zn 2+ And mixing and crosslinking the aqueous solution of the metal ions to obtain the sodium alginate medicine-carrying gel microcapsule, wherein the volume ratio of the dispersion liquid of the bioactive macromolecular medicine-sodium alginate to the aqueous solution of the metal ions is 1 (0.1-10).
In the present invention, there is no particular limitation on the emulsification method, the instillation method or the spraying method, and methods and operations or reaction conditions conventional in the art may be employed as long as crosslinking of sodium alginate with metal ions can be achieved; for example, in some specific examples, the bioactive macromolecular drug-sodium alginate dispersion is emulsified with 0.5wt% to 3wt% Ca 2+ Or Zn 2+ The specific method for the mixed crosslinking of the aqueous solution of the plasma metal ions is as follows:
60ml of liquid paraffin, 12ml of Span 80 and 3ml of Tween 80 are uniformly mixed to obtain clear and transparent light yellow liquid which is used as a mixed oil phase. At 800rpm, 10ml of sodium alginate solution containing 10mg of catalase was slowly dropped into the mixed oil phase by a syringe, the rotation speed was adjusted up to 1000rpm, and the emulsion was emulsified for 30min to a white emulsion. Next, 10ml of CaCl was slowly dropped thereinto 2 After stirring the solution for a further 20min, 30ml of cyclopentane (CaCl) were added thereto 2 The volume ratio of the aqueous solution to the cyclopentane is 1:3) to break the emulsion system, so as to separate the microcapsule sediment, and after 10000rpm freeze centrifugation, the microcapsule is washed with isopropanol for 2-3 times to obtain the calcium alginate drug-carrying microcapsule.
According to the invention, in the step N, the sodium alginate medicine-carrying gel microcapsule is dispersed into a solution of chitosan grafted with a digestive enzyme inhibitor, the solution is incubated at room temperature, the chitosan grafted with the digestive enzyme inhibitor is adsorbed on the surface of the sodium alginate medicine-carrying gel microcapsule through electrostatic action, and the gel microcapsule system for orally delivering the bioactive macromolecular medicine is obtained through filtration, washing and drying.
Specifically, in step N, the method for preparing chitosan grafted with a digestive enzyme inhibitor comprises:
(1) The chitosan grafted with the digestive enzyme inhibitor is dissolved in acetic acid-sodium acetate buffer solution (concentration 0.1-0.2M) with ph=4-6, so that the final mass fraction of the chitosan grafted with the digestive enzyme inhibitor is 0.1wt% -2 wt%.
(2) Dispersing the sodium alginate drug-loaded gel microcapsule into the solution of the chitosan grafted with the digestive enzyme inhibitor according to the ratio of the mass (g) of the sodium alginate drug-loaded gel microcapsule to the volume (mL) of the chitosan solution of 1 (1-10) g/mL, incubating for 5-60min at room temperature, adsorbing the chitosan grafted with the digestive enzyme inhibitor onto the surface of the sodium alginate drug-loaded gel microcapsule through electrostatic action, filtering, washing and drying to obtain the gel microcapsule system for oral delivery of the bioactive macromolecular drug.
III. Examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
0.3wt% chitosan solution was prepared, pH=6.0 was adjusted, methyl gallate was added thereto to 10mM (mass ratio of phenolic precursor to chitosan: 1:1.63), aprotinin was added thereto to 1mM (mass ratio of digestive enzyme inhibitor to chitosan: 2.17:1), tyrosinase was added thereto to 60U/mL (enzyme activity unit of polyphenol oxidase (U): molar precursor: 6000000:1) after stirring uniformly, and stirring was carried out at room temperature for 12 hours. The obtained solution was packed into a 10kDa dialysis bag and dialyzed against acetic acid-sodium acetate buffer (0.1M, pH=5.8) for 24 hours to remove excess methyl gallate and free aprotinin, thereby obtaining aprotinin-grafted chitosan solution (CS-AP).
0.2g of sodium alginate was dissolved in 10mL of deionized water to prepare a sodium alginate solution having a concentration of 2wt%. Weighing 10mg of catalase, dissolving in prepared sodium alginate solution (catalase content is 5 wt%) to form catalase-sodium alginate dispersion liquid, and emulsifying with 2wt% of CaCl 2 Mixing and crosslinking the aqueous solution to obtain the sodium alginate medicine-carrying gel microcapsule.
The specific mode is that 60ml of liquid paraffin, 12ml of Span 80 and 3ml of Tween 80 are uniformly mixed to obtain clear and transparent light yellow liquid which is used as a mixed oil phase. At 800rpm, 10ml of a dispersion containing 10mg of catalase-sodium alginate was slowly dropped into the mixed oil phase by a syringe, the rotation speed was adjusted up to 1000rpm, and the emulsion was emulsified for 30min to a white emulsion. Next, 10ml of CaCl was slowly dropped thereinto 2 Aqueous solution (catalase-sodium alginate dispersion and CaCl) 2 The volume ratio of the aqueous solution was 1:1), stirring was continued for 20min, and 30ml of cyclopentane (CaCl) was added thereto 2 The volume ratio of the aqueous solution to the cyclopentane is 1:3) to break the emulsion system, so as to separate the microcapsule sediment, and after 10000rpm freeze centrifugation, the microcapsule is washed with isopropanol for 2-3 times to obtain the calcium alginate drug-carrying microcapsule.
Dissolving 0.06g of chitosan grafted with aprotinin in 20mL of 0.2M acetic acid-sodium acetate solution with pH=5.0 to prepare 0.3wt% of chitosan solution grafted with aprotinin, soaking the obtained calcium alginate drug-loaded microcapsule in the chitosan solution (the mass volume ratio of the sodium alginate drug-loaded gel microcapsule to the solution of the chitosan grafted with the digestive enzyme inhibitor is 1:2), incubating for 15min, centrifuging, washing with deionized water for 2-3 times, and freeze-drying to obtain the chitosan-sodium alginate drug-loaded microcapsule with the aprotinin grafted on the surface.
Example 2:
the surface morphology of the microcapsules after modification was photographed by a Scanning Electron Microscope (SEM), and the results are shown in fig. 2. The liquid obtained by dialysis was lyophilized to obtain aprotinin-grafted chitosan, the graft modification of which was characterized by fourier infrared spectroscopy (FT-IR) and the results are shown in fig. 3.
From fig. 2, the surface of the modified microcapsule is changed from sharp folds to a compact encapsulation layer, which shows that the encapsulation effect of chitosan is changed to a certain extent after modification, thus proving that the modification is successful.
As can be seen from fig. 3, in the infrared spectrum of the aprotinin-grafted chitosan, the peak of the amide bond appears clearly, and thus it can be demonstrated that aprotinin was successfully grafted to chitosan.
Example 3:
taking a certain amount of microcapsules, respectively treating with artificial gastric juice and artificial intestinal juice according to gradients for different times. Following centrifugation, an equal amount of carbonate buffer at ph=8.0 was added, and after complete dissolution of the microcapsules, the supernatant was centrifuged and the enzyme activity retention was measured using a catalase activity assay kit, and the results are shown in fig. 4.
As can be seen from fig. 4, the modified microsphere still has a good gastric acid resistance effect, but in the aspect of the enzymolysis resistance effect, the enzymolysis resistance effect of the modified microsphere is obviously improved, about 80% of enzyme activity can be reserved in 4 hours, and the unmodified microsphere is inactive in 1 hour.
Example 4
By combining the experiments, the sodium alginate/chitosan microcapsule can better resist the influence of gastric acid, and the microcapsule still has a defect in resisting trypsin degradation. Therefore, the effect of the addition of various adjuvants on the resistance of the microcapsules to trypsin degradation was investigated in this section. And a certain amount of uric acid was dissolved in Tris-HCl buffer (0.01M, ph=8.5) to give a final concentration of 0.05M. And (3) putting a certain amount of microcapsules into the prepared uric acid solution, continuously stirring for 12 hours at 37 ℃, taking 100 mu L of the solution as a sample at intervals, measuring the absorbance at 293nm by using a NanoPhotometer-N60, and calculating the corresponding uric acid concentration according to a standard curve.
Example 5
A 0.5wt% chitosan solution was prepared, ph=6.0 was adjusted, gallic acid was added thereto to 10mM (mass ratio of phenolic precursor to chitosan 1:2.94), aprotinin was added to 1mM (mass ratio of digestive enzyme inhibitor to chitosan 1.31:1), tyrosinase was added to 80U/mL (enzyme activity unit of polyphenol oxidase (U): phenolic precursor molar number=8000000:1) after stirring uniformly, and stirring was carried out at room temperature for 12 hours. The obtained solution was packed into a 10kDa dialysis bag and dialyzed against acetic acid-sodium acetate buffer (0.1M, pH=5.8) for 24 hours to remove excess methyl gallate and free aprotinin, thereby obtaining aprotinin-grafted chitosan solution (CS-AP).
0.2g of sodium alginate was dissolved in 10mL of deionized water to prepare a sodium alginate solution having a concentration of 2wt%. Weighing 10mg of catalase, dissolving in prepared sodium alginate solution (catalase content is 5 wt%) to form catalase-sodium alginate dispersion liquid, and emulsifying with 2wt% of CaCl 2 Mixing and crosslinking the aqueous solution to obtain the sodium alginate medicine-carrying gel microcapsule.
The specific mode is that 60ml of liquid paraffin, 12ml of Span 80 and 3ml of Tween 80 are uniformly mixed to obtain clear and transparent light yellow liquid which is used as a mixed oil phase. At 800rpm, 10ml of sodium alginate solution containing 10mg of catalase was slowly dropped into the mixed oil phase by a syringe, the rotation speed was adjusted up to 1000rpm, and the emulsion was emulsified for 30min to a white emulsion. Next, 15ml of CaCl was slowly dropped thereinto 2 Aqueous solution (catalase-sodium alginate dispersion and CaCl) 2 The volume ratio of the aqueous solution was 1:1.5), and after stirring was continued for 20min, 30ml of cyclopentane (CaCl) was added thereto 2 The volume ratio of aqueous solution to cyclopentane was 1:3) to break the emulsion system, precipitate the microcapsules, freeze centrifuge at 10000rpm, wash with isopropanolWashing for 2-3 times to obtain the calcium alginate drug-carrying microcapsule.
Dissolving 0.5g of chitosan grafted with aprotinin in 100mL of 0.2M acetic acid-sodium acetate solution with pH=5.0 to prepare 0.5wt% of chitosan solution grafted with aprotinin, soaking the obtained microcapsule in the chitosan solution (the mass volume ratio of the sodium alginate drug-loaded gel microcapsule to the solution of the chitosan grafted with the digestive enzyme inhibitor is 1:10), incubating for 15min, centrifuging, washing with deionized water for 2-3 times, and freeze-drying to obtain the sodium alginate-chitosan drug-loaded microcapsule of the surface cross-linked aprotinin.
From the above examples, it can be seen that aprotinin can be successfully grafted onto chitosan under the street of a phenol precursor by the catalysis of polyphenol oxidase, and the anti-enzymolysis effect of the sodium alginate microcapsule is greatly improved after the encapsulation of the aprotinin grafted chitosan.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, not to be construed as limiting the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (10)
1. An enteric adhesive enzymolysis resistant gel microcapsule oral delivery system for bioactive macromolecular drugs, comprising:
the chitosan is adsorbed on the surface of the sodium alginate drug-loaded gel microcapsule and grafted with digestive enzyme inhibitor;
the sodium alginate medicine-carrying gel microcapsule is composed of a sodium alginate wall material and bioactive macromolecular medicines embedded in the sodium alginate wall material.
2. The oral delivery system of claim 1, wherein the oral delivery system comprises,
the sodium alginate wall material is formed by crosslinking a sodium alginate solution and metal ions; preferably, the metal ions include Ca 2+ And/or Zn 2+ ；
And/or the digestive enzyme inhibitor is a substance having an activity of inhibiting human gastrointestinal protease; preferably, the digestive enzyme inhibitor comprises one or more of aprotinin, soybean trypsin inhibitor, chymotrypsin and pepsin inhibitor;
and/or, the bioactive macromolecular drug comprises nucleic acid, polypeptide and protein; preferably, the bioactive macromolecular drug comprises one or more of GLP-1 and analogues thereof, insulin, glucagon, sialin-4, salmon calcitonin, interferon, various antibodies, trypsin, thrombin, lysozyme, catalase, protease inhibitors and catalase.
3. The oral delivery system of claim 1, wherein the oral delivery system comprises,
the chitosan grafted with the digestive enzyme inhibitor is formed by using a phenol precursor as a crosslinking medium to crosslink and fix the digestive enzyme inhibitor on the chitosan under the catalysis of polyphenol oxidase;
preferably, the polyphenol oxidase is an enzyme capable of oxidizing phenol or polyphenol by molecular oxygen to form the corresponding quinone; further preferably, the polyphenol oxidase comprises monophenol monooxidase (tyrosinase), bisphenol oxidase (catechol oxidase) and laccase;
and/or the phenolic precursor is a natural substance having a phenolic hydroxyl group; further preferably, the phenolic precursor comprises methyl gallate, gallic acid, chlorogenic acid, arbutin, caffeic acid, p-benzoquinone, catechol.
4. A method of preparing an enteric adhesive enzymolysis resistant gel microcapsule oral delivery system for a bioactive macromolecular drug, comprising:
step L, under the catalysis of polyphenol oxidase, using a phenol precursor as a crosslinking medium to crosslink and fix a digestive enzyme inhibitor on chitosan, so as to obtain chitosan grafted with the digestive enzyme inhibitor;
step M, embedding bioactive macromolecular drugs into the sodium alginate wall material to obtain sodium alginate drug-loaded gel microcapsules;
and step N, coating chitosan grafted with a digestive enzyme inhibitor on the surface of the sodium alginate drug-loaded gel microcapsule to obtain the intestinal adhesion anti-enzymolysis gel microcapsule oral delivery system of the bioactive macromolecular drug.
5. The method according to claim 4, wherein in step L, the phenol precursor and the digestive enzyme inhibitor are dissolved in a chitosan solution, and the mixture is added with polyphenol oxidase, incubated at room temperature, dialyzed, filtered, and freeze-dried to obtain the chitosan grafted with the digestive enzyme inhibitor.
6. The method according to claim 5, wherein,
the chitosan solution is formed by dissolving chitosan in 0.1M hydrochloric acid solution, and adjusting pH=5.5-6.5 by 0.01M NaOH solution; preferably, the concentration of the chitosan solution is 0.1-2 wt%;
and/or, in the step L, the mass ratio of the phenolic precursor to the chitosan is 1 (1-10);
and/or the mass ratio of the digestive enzyme inhibitor to the chitosan is 5:1-1:2;
and/or, the enzyme activity unit (U) of polyphenol oxidase: phenolic precursor mole number= (1000000 ～ 8000000): 1;
and/or, the incubation time at room temperature is 6-24h.
7. The preparation method of claim 4, wherein in the step M, the bioactive macromolecular drug is dispersed in sodium alginate solution to form bioactive macromolecular drug-sodium alginate dispersion, and the bioactive macromolecular drug-sodium alginate dispersion is mixed and crosslinked with metal ion solution through processing and molding treatment to obtain the sodium alginate drug-loaded gel microcapsule; preferably, the method of the process molding treatment includes an emulsification method, a instillation method or a spraying method.
8. The method according to claim 7, wherein,
the sodium alginate solution is formed by dissolving sodium alginate in water; preferably, the concentration of the sodium alginate solution is 0.5-5 wt%;
and/or the content of the bioactive macromolecular drug in the bioactive macromolecular drug-sodium alginate dispersion liquid is 0.5-40 wt%;
and/or the concentration of the metal ion aqueous solution is 0.5-3 wt%;
and/or the volume ratio of the bioactive macromolecular drug-sodium alginate dispersion liquid to the metal ion aqueous solution is 1 (0.1-10).
9. The preparation method according to claim 4, wherein in the step N, the sodium alginate drug-loaded gel microcapsule is dispersed in a solution of chitosan grafted with a digestive enzyme inhibitor, incubated at room temperature, the chitosan grafted with the digestive enzyme inhibitor is adsorbed on the surface of the sodium alginate drug-loaded gel microcapsule by electrostatic action, and the gel microcapsule system for oral delivery of bioactive macromolecular drugs is obtained by filtration, washing and drying.
10. The method according to claim 9, wherein,
in the step N, the solution of the chitosan grafted with the digestive enzyme inhibitor is formed by dissolving the chitosan grafted with the digestive enzyme inhibitor in an acetic acid-sodium acetate buffer solution with ph=4-6; preferably, the content of the chitosan grafted with the digestive enzyme inhibitor in the solution of the chitosan grafted with the digestive enzyme inhibitor is 0.1 to 2 weight percent; and/or the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.2M;
and/or the mass volume ratio of the sodium alginate medicine-carrying gel microcapsule to the solution of the chitosan grafted with the digestive enzyme inhibitor is 1 (1-10) g/mL;
and/or, in the step N, the incubation time at room temperature is 5-60min.
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