CN117919434A - Self-frame nano drug delivery system and preparation method and application thereof - Google Patents
Self-frame nano drug delivery system and preparation method and application thereof Download PDFInfo
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- CN117919434A CN117919434A CN202410108790.9A CN202410108790A CN117919434A CN 117919434 A CN117919434 A CN 117919434A CN 202410108790 A CN202410108790 A CN 202410108790A CN 117919434 A CN117919434 A CN 117919434A
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- quercetin
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Abstract
The invention relates to the technical field of medical nano materials, in particular to a self-frame nano drug delivery system, a preparation method and application thereof, and the preparation method of the self-frame nano drug delivery system comprises the following steps: dissolving natural polyphenol, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers in a solvent to obtain a mixed solution; wherein the mol ratio of the mixture of natural polyphenol and functional molecule containing diselenide bond to hexachlorocyclotriphosphazene monomer is 1-3:1; mixing the mixed solution with an acid binding agent, and carrying out polymerization reaction under the catalysis of the acid binding agent to prepare the self-frame nano drug delivery system. The self-frame nanometer drug delivery system prepared by the invention is applied to the application of preparing the drug for treating Inflammatory Bowel Disease (IBD), can prolong the half life of the drug, realize ROS responsive degradation and sustained release of natural polyphenol compounds, and solve the problems of poor structural stability, low drug availability and the like of the existing IBD nanometer drug delivery system.
Description
Technical Field
The invention relates to the technical field of medical nano materials, in particular to a self-frame nano drug delivery system and a preparation method and application thereof.
Background
Inflammatory bowel disease, IBD for short, is a common chronic gastrointestinal inflammation that can cause intestinal microbial imbalance, impaired intestinal mucosal barrier, and reduced immune function. Currently, the clinical line mainly uses drugs such as aminosalicylic acid (5-ASA), antibiotics, corticosteroids and the like to reduce inflammation and repair intestinal barriers. However, the clinically used drugs cannot form specific targeted therapy on the colon, so that the drugs cannot be effectively delivered to an inflammation area, the curative effect of the drugs is reduced, and adverse reactions can be generated on other organs. Therefore, there is a need to develop new drugs to reduce the loss of the drug in the upper digestive tract and increase the absorption in the inflammatory areas of the intestine.
Notably, overproduction of Reactive Oxygen Species (ROS), e.g., increased by a factor of 10-100, is an important pathological feature of IBD and is a direct cause of signal transduction disorders, inflammatory reactions, and DNA damage. Excessive accumulation of ROS is a central link in the pathological process of IBD, directly accelerating the development and progression of the disease. Therefore, scavenging ROS has become an effective treatment strategy for IBD.
The nano drug delivery system can enhance the permeability and detention effect in intestinal mucosa and is developed and applied step by step due to the advantages of small volume and high permeability, thus bringing new ideas for preventing and treating various diseases. However, in oral administration to treat IBD, digestive fluids can lead to rapid degradation of the nano-delivery system and diarrhea caused by IBD also leads to rapid drug clearance. Although various nano-drug delivery systems have been developed to treat IBD, there are still problems of insufficient residence time, low targeting efficiency to the inflamed colon, and the complex synthetic process also limits its clinical application.
Disclosure of Invention
In order to solve the problems of poor structural stability, low drug availability and the like of the existing IBD nano drug delivery system, the invention aims to provide a self-frame nano drug delivery system and a preparation method and application thereof.
According to the invention, the hexafunctional cyclotriphosphazene monomer and the functional molecule containing polyphenol hydroxyl and diselenide bond are crosslinked and polymerized to prepare the oral nano-drug which has uniform particle size, biocompatibility, high stability, antioxidation and anti-inflammatory property and is superior to the clinical first-line drug aminosalicylic acid, so that the half life of the drug can be prolonged, the ROS (reactive oxygen species) is degraded in response and the natural polyphenol compound is continuously released, toxic byproducts are not generated, the defect of the existing oral drug delivery system in the application field is effectively solved, and the types of IBD drug delivery systems are enriched.
In order to achieve the above object, the technical scheme of the present invention is as follows.
The first aspect of the present invention provides a method for preparing a self-framing nano-drug delivery system, comprising the steps of:
dissolving natural polyphenol, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers in a solvent to obtain a mixed solution; wherein the molar ratio of the sum of the amount of the natural polyphenol substance and the amount of the diselenide bond-containing functional molecule substance to the hexachlorocyclotriphosphazene monomer is 1-3:1;
mixing the mixed solution with an acid binding agent, and reacting under the catalysis of the acid binding agent to prepare the self-frame nano drug delivery system.
In a preferred embodiment, the natural polyphenol is any one of quercetin, resveratrol and curcumin.
In a preferred embodiment, the diselenide bond-containing functional molecule is any one of selenocyamine and 4,4' -diselenidenediyldianiline.
In a preferred embodiment, the molar ratio of natural polyphenols to functional molecules containing diselenide linkages is from 1:1 to 2.
In a preferred embodiment, the acid binding agent is any one of triethylamine and sodium hydroxide solution.
In a preferred embodiment, the molar concentration of the sodium hydroxide solution is between 0.05 and 0.125mol/L.
In a preferred embodiment, the amount of acid-binding agent is 1 to 5% of the total volume of the mixed solution.
In a preferred embodiment, the solvent is acetonitrile or tetrahydrofuran.
In a preferred embodiment, the conditions under which the reaction takes place under catalysis of the acid-binding agent are:
the reaction temperature is 0-40 ℃; the reaction time is 3-5 h.
In a preferred embodiment, the reaction is completed and then further comprises centrifugation and drying; wherein the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10min.
In a second aspect, the present invention provides a self-framed nano-drug delivery system prepared by the preparation method of the first aspect.
In a preferred embodiment, the self-framed nano drug delivery system is prepared from natural polyphenol, a functional molecule containing diselenide bond and a hexachlorocyclotriphosphazene monomer, wherein the Hexachlorocyclotriphosphazene (HCCP) monomer contains six P-Cl bonds with lower bond energy, can be subjected to nucleophilic substitution reaction with the natural polyphenol containing phenolic hydroxyl groups and the diselenide bond functional molecule containing polyamino groups under the catalysis of an acid binding agent, and is polymerized to form the self-framed nano drug delivery system.
In a preferred embodiment, the self-framing nano drug delivery system is prepared by cross-linking polymerization of hexachlorocyclotriphosphazene monomer, natural polyphenol containing polyphenol hydroxyl and functional molecule containing diselenide bond;
The self-frame nanometer drug delivery system is prepared by taking hexachlorocyclotriphosphazene as the center of a crosslinking structure and utilizing P-Cl bond of hexachlorocyclotriphosphazene and phenolic hydroxyl of natural polyphenol and/or amino of functional molecules containing diselenide bond to undergo nucleophilic substitution reaction under the catalysis of an acid binding agent.
In a third aspect, the present invention provides a use of a self-framing nano-delivery system for the preparation of a medicament for the treatment of inflammatory bowel disease, the self-framing nano-delivery system being as described in the second aspect.
In a preferred embodiment, the medicament is an oral medicament.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention uses hexa-functional cyclotriphosphazene monomer, natural polyphenol containing polyphenol hydroxyl and functional molecule containing diselenide bond to crosslink and polymerize, prepares the oral nanometer medicine which has uniform particle diameter, biocompatibility, high stability, antioxidation, anti-inflammatory and better than the clinical first-line medicine aminosalicylic acid, solves the problems of poor structural stability, single treatment function, low medicine availability and the like of the traditional nanometer medicine delivery system, and enriches the types of medicine delivery systems for treating inflammatory intestines.
2. The self-frame nano drug delivery system prepared by the invention is a monodisperse microsphere, has good water dispersion stability and physiological stability, and can be stored for a long time; can also resist the extreme environment of the gastrointestinal tract, and can be effectively degraded in the inflammatory colon environment. The negative surface charge and tissue adhesiveness of the composition can realize long-term enrichment at inflammatory colon parts, and frame degradation and sustained release of natural polyphenol can be realized by responding to and eliminating overloaded ROS; can safely and effectively treat inflammatory bowel disease.
Drawings
FIG. 1A is a transmission electron microscope image of a self-framing nano-drug delivery system of quercetin prepared in example 1; FIG. B is a transmission electron microscope image of the resveratrol self-framing nano-delivery system prepared in example 2; panel C is a transmission electron microscope image of the curcumin self-framing nano-delivery system prepared in example 3.
Figure 2 is a graph of the hydrated diameter of quercetin self-framing nano-delivery system prepared in example 1.
Figure 3 is a surface potential map of quercetin self-framing nano-delivery system prepared in example 1.
FIG. 4 is an infrared spectrum of a quercetin self-framing nano-delivery system prepared in example 1.
Fig. 5 is a graph of drug loading and encapsulation rate of quercetin self-framing nano-delivery system prepared in example 1.
Figure 6 is a graph of the water dispersibility of quercetin self-framing nano-delivery system prepared in example 1.
FIG. 7 is a graph of the physiological stability of quercetin self-framing nano-delivery system prepared in example 1.
Figure 8 is a graph of oral drug degradability of quercetin prepared in example 1in a simulated gastrointestinal environment from a framed nano-drug delivery system.
Figure 9 is a graph of ROS capture performance of quercetin self-framing nano-delivery systems prepared in example 1.
Fig. 10 is an in vivo overall ROS level of quercetin self-framed nano-delivery system prepared in example 1 (a) and corresponding quantification graph (B). Wherein, the B graph is a quantitative graph of the A graph.
FIG. 11 is a graph of the therapeutic effect of quercetin self-framing nano-delivery system prepared in example 1.
FIG. 12 is a graph of the ROS level in colon tissue and colon permeability of a quercetin self-framing nano-delivery system prepared in example 1.
Figure 13 is a safety profile of quercetin self-framing nano-delivery system prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The natural polyphenol is a bioactive compound widely derived from plant kingdom, such as quercetin, resveratrol, curcumin, etc., and has antioxidant, antiinflammatory, antibacterial, antitumor, antiviral, blood sugar reducing, blood pressure lowering, immunity regulating, cardiovascular protecting, etc. therapeutic effects. However, natural polyphenol compounds have obvious defects in practical application, such as low solubility, poor stability, short half-life, rapid metabolism, low gastrointestinal absorption, limited bioavailability and the like, and severely limit the deep application of the natural polyphenol compounds in the fields of foods and medicines.
Selenium (Se) is a trace nonmetallic element necessary for maintaining normal physiological functions and structures of human bodies, and researches prove that small molecules or bioactive materials containing the Se element not only can realize drug release while removing excessive ROS in inflammatory microenvironment, but also can realize wide biological functions through responding and activating multiple ways of inducing apoptosis such as endogenous ROS production related signal channels, DNA cleavage, caspase-3 activation, tumor suppressor protein p53 increase, inactivated Protein Kinase C (PKC), mitochondrial injury and the like. In addition, the selenium-containing micromolecules can be used as a supplement to effectively regulate and control oxidative stress, play a role in resisting oxidization and protecting injury with concentration selectivity, and are expected to become an ideal effective component of the IBD prevention and treatment functional medicine.
Based on the method, the hexa-functional cyclotriphosphazene monomer, the natural polyphenol containing polyphenol hydroxyl and the functional molecule containing diselenide bond are crosslinked and polymerized to prepare the oral nanometer medicament of the aminosalicylic acid (5-ASA) which has uniform particle size, biocompatibility, high stability, antioxidation and anti-inflammatory property and is superior to the clinical first-line medicament, the problems of poor structural stability, single treatment function, low medicament availability and the like of the traditional nanometer medicament delivery system are solved, and the medicament delivery system type for treating inflammatory bowel is enriched.
The first aspect of the present invention provides a method for preparing a self-framing nano-drug delivery system, comprising the steps of:
dissolving natural polyphenol, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers in a solvent to obtain a mixed solution; wherein the molar ratio of the sum of the amount of the natural polyphenol substance and the amount of the diselenide bond-containing functional molecule substance to the hexachlorocyclotriphosphazene monomer is 1-3:1;
Mixing the mixed solution with an acid binding agent, and carrying out polymerization reaction under the catalysis of the acid binding agent to prepare the self-frame nano drug delivery system.
In a preferred embodiment, the natural polyphenol is any one of quercetin, resveratrol and curcumin.
In a preferred embodiment, the diselenide bond-containing functional molecule is any one of selenocyamine and 4,4' -diselenidenediyldianiline.
In a preferred embodiment, the molar ratio of the natural polyphenol compound to the diselenide bond-containing functional molecule is from 1:1 to 2.
In a preferred embodiment, the acid binding agent is any one of triethylamine and sodium hydroxide solution.
In a preferred embodiment, the concentration of the sodium hydroxide solution is between 0.05 and 0.125mol/L.
The concentration of sodium hydroxide is too low or too high to easily influence the morphology, particle size, dispersity and the like of the nano-drug, and the proper concentration of sodium hydroxide is favorable for generating regular morphology, uniform particle size and mono-disperse sphericity, thereby being favorable for improving the drug delivery efficiency and human body absorption.
In a preferred embodiment, the amount of acid-binding agent is 1 to 5% of the total volume of the mixed solution.
In a preferred embodiment, the solvent is acetonitrile or tetrahydrofuran.
In a preferred embodiment, the conditions under which the reaction takes place under catalysis of the acid-binding agent are:
The reaction temperature is 0-40 ℃; the reaction time is 3-5 h; examples thereof include a reaction at 0℃for 5 hours, a reaction at 25℃for 5 hours, a reaction at 40℃for 5 hours, a reaction at 0℃for 3 hours, a reaction at 25℃for 3 hours, a reaction at 40℃for 3 hours, a reaction at 0℃for 4 hours, a reaction at 25℃for 4 hours, and a reaction at 40℃for 4 hours.
In a preferred embodiment, the reaction is completed and then further comprises centrifugation and drying; wherein the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10min. Examples of the centrifugal force include centrifugation at 10000rpm for 10min, centrifugation at 9000rpm for 10min, and centrifugation at 8000rpm for 10min. In a preferred embodiment, the drying is freeze-drying at a temperature of-50℃for a drying time of 20-24 hours. Examples include freeze-drying at-50℃for 20 hours and freeze-drying at-50℃for 24 hours.
In a second aspect, the present invention provides a self-framed nano-drug delivery system prepared by the preparation method of the first aspect.
In a preferred embodiment, the self-framed nano drug delivery system is composed of natural polyphenol, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene, wherein Hexachlorocyclotriphosphazene (HCCP) monomer contains P-Cl bonds with lower bond energy, and can be subjected to nucleophilic substitution reaction with the natural polyphenol containing phenolic hydroxyl groups and the diselenide functional molecules containing polyamino groups under the catalysis of an acid binding agent at will, so that the self-framed nano drug delivery system is formed by polymerization.
In a third aspect, the present invention provides a use of a self-framing nano-delivery system for the preparation of a medicament for the treatment of inflammatory bowel disease, the self-framing nano-delivery system being as described in the second aspect.
In a preferred embodiment, the medicament is an oral medicament.
In a preferred embodiment, the oral medicament is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the self-framing nano-delivery system according to the second aspect, and the pharmaceutically acceptable excipients include fillers, disintegrants, binders and lubricants.
In a preferred embodiment, the filler is at least one of mannitol, lactose, microcrystalline cellulose, or starch; the disintegrating agent is at least one of carboxymethyl starch sodium, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethyl cellulose, hydroxypropyl starch and low-substituted hydroxypropyl cellulose; the adhesive is at least one of povidone K30, ethyl cellulose and hydroxypropyl cellulose; the lubricant is at least one of magnesium stearate and sodium dodecyl sulfate. Of course, if necessary, a disintegrating aid (e.g., silica gel micropowder), a wetting agent (e.g., water), and the like may be added.
In a preferred embodiment, the pharmaceutically acceptable excipients further comprise a flavoring agent for improving the taste of the oral formulation by adding an appropriate amount of the flavoring agent. Of course, a sugar coating layer can also be coated on the surface of the tablet core.
In a preferred embodiment, the oral medicament is a solid oral formulation, including tablets, capsules, granules. The self-framing nano-delivery system according to the second aspect and pharmaceutically acceptable excipients can be readily prepared by those skilled in the art into conventional solid oral dosage forms, such as tablets, capsules or granules, according to conventional techniques in the formulation arts.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
The methods described in the examples below are conventional, unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
Wherein, quercetin is delivered from the frame nanometer drug delivery system, which is marked as HQuSe; ROS, reactive oxygen species.
Example 1
A preparation method of a quercetin self-frame nano drug delivery system comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound (100W) (400 rpm) to completely dissolve, to obtain a mixed solution.
10ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 2
A preparation method of a resveratrol self-frame nano drug delivery system comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.415g (1.82 mmol) of resveratrol (Rv) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out in 500mL of acetonitrile, and stirred with the aid of ultrasound (100W) (400 rpm) to be completely dissolved, to obtain a mixed solution.
10ML of Triethylamine (TEA) was measured and added to the above mixed solution, the mixture was reacted at 25℃for 5 hours, after the completion of the reaction, the precipitate was collected by high-speed centrifugation (10000 rpm,10 minutes), the product was washed three times with ethanol and ultrapure water, respectively, and finally, the product was uniformly dispersed in 10mL of ultrapure water and stored, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, cooling to room temperature, taking out, and continuing to freeze-dry for 24 hours to obtain the resveratrol self-frame nano drug delivery system.
Example 3
A method for preparing a curcumin self-frame nano drug delivery system, which comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.67g (1.82 mmol) of curcumin (Cur) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) are weighed out and dissolved in 500mL of acetonitrile, and the mixture solution is obtained by stirring (400 rpm) with the aid of ultrasound (100W).
10ML of Triethylamine (TEA) was measured and added to the above mixed solution, the mixture was reacted at 25℃for 5 hours, after the completion of the reaction, the precipitate was collected by high-speed centrifugation (10000 rpm,10 minutes), the product was washed three times with ethanol and ultrapure water, respectively, and finally, the product was uniformly dispersed in 10mL of ultrapure water and stored, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuing to freeze-dry for 24 hours to obtain the curcumin self-frame nano drug delivery system.
Example 4
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that the molar ratio of a functional molecule containing a diselenide bond to a hexachlorocyclotriphosphazene monomer is 2:1, and the preparation method specifically comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 1.17g (3.64 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound (100W) (400 rpm) to obtain a mixed solution.
10ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 5
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that the molar ratio of a functional molecule containing a diselenide bond to a hexachlorocyclotriphosphazene monomer is 3:1, and the preparation method specifically comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 1.755g (5.46 mmol) of selenocysteine (Se-Se) are weighed, dissolved in 500mL of acetonitrile and stirred completely by ultrasound (100W) at 400rpm to obtain a mixed solution.
10ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 6
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that a functional molecule containing a diselenide bond is 4,4' -diselenide diyldiphenylamine, and the preparation method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.623g (1.82 mmol) of 4,4' -diselenidenediyldianiline are weighed out, dissolved in 500mL of acetonitrile and stirred completely with the aid of ultrasound (100W) (400 rpm) to give a mixed solution.
10ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 7
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that an acid binding agent is sodium hydroxide solution with concentration of 0.05-0.125 mol/L, and the preparation method specifically comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.623g (1.82 mmol) of 4,4' -diselenidenediyldianiline are weighed out, dissolved in 500mL of acetonitrile and stirred completely with the aid of ultrasound (100W) (400 rpm) to give a mixed solution.
10ML of sodium hydroxide solution with the concentration of 0.05-0.125 mol/L is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the high-speed centrifugation (10000 rpm,10 min) is carried out to collect the precipitate; the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
The reaction is carried out on 0.05mol/L, 0.75mol/L and 0.125mol/L of sodium hydroxide solution with different concentrations respectively, and the product prepared in the concentration range of 0.05-0.125 mol/L is relatively regular in morphology and uniform in particle size, and is in a monodisperse spherical shape. And when the concentration is too high or too low, the morphology, the particle size and the dispersity of the nano-drug are easily affected.
Example 8
A method for preparing a quercetin self-framing nano drug delivery system is different from example 1 in that the amount of an acid binding agent is 1% of the total volume of a mixed solution. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 1.82mmol of selenocysteine (Se-Se) are weighed, dissolved in 500mL of acetonitrile, and stirred (400 rpm) with the aid of ultrasound 100W to completely dissolve, to obtain a mixed solution.
Weighing 5mL of Triethylamine (TEA), adding the mixture into the mixed solution, reacting for 5 hours at 25 ℃, centrifuging at a high speed (10000 rpm,10 min) after the reaction is finished, and collecting a precipitate; the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 9
A method for preparing a quercetin self-framing nano drug delivery system is different from example 1 in that the amount of acid binding agent is 3% of the total volume of the mixed solution. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound 100W (400 rpm) to give a mixed solution.
15ML of Triethylamine (TEA) was measured and added to the above mixed solution, the mixture was reacted at 25℃for 5 hours, and after the reaction was completed, the precipitate was collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 10
A method for preparing a quercetin self-framing nano drug delivery system is different from example 1 in that the amount of acid binding agent is 4% of the total volume of the mixed solution. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound 100W (400 rpm) to give a mixed solution.
20ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 11
A method for preparing a quercetin self-framing nano drug delivery system is different from example 1 in that the amount of acid binding agent is 5% of the total volume of the mixed solution. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound 100W (400 rpm) to give a mixed solution.
25ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 12
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that the reaction temperature is 0 ℃ and the reaction time is 5 hours. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound 100W (400 rpm) to give a mixed solution.
25ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at the temperature of 0 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Example 13
A preparation method of a quercetin self-frame nano drug delivery system is different from example 1 in that the reaction temperature is 40 ℃ and the reaction time is 3 hours. The specific method comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) and 0.585g (1.82 mmol) of selenocysteine (Se-Se) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound 100W (400 rpm) to give a mixed solution.
25ML of Triethylamine (TEA) was added to the above mixed solution and reacted at 40℃for 3 hours, after the reaction was completed, the mixture was centrifuged at a high speed (8000 rpm,10 min) to collect a precipitate; the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system which is marked as HQuSe.
Comparing the products prepared in examples 1, 12 and 13, it was found that the particle sizes of the products prepared in examples 1, 12 and 13 were relatively close, indicating that the different temperatures had little effect on the particle size of the products.
Comparative example 1
The preparation method of the quercetin nanometer drug delivery system without the diselenide bond functional molecule comprises the following steps:
0.632g (1.82 mmol) of Hexachlorocyclotriphosphazene (HCCP), 0.55g (1.82 mmol) of quercetin (Qu) were weighed out, dissolved in 500mL of acetonitrile, and stirred with the aid of ultrasound (100W) (400 rpm) to completely dissolve, thereby obtaining a mixed solution.
10ML of Triethylamine (TEA) is measured and added into the mixed solution to react for 5 hours at 25 ℃, and after the reaction is finished, the precipitate is collected by high-speed centrifugation (10000 rpm,10 min); the product was washed three times with ethanol and ultrapure water, respectively, and finally, uniformly dispersed into 10mL of ultrapure water for preservation, to obtain a dispersion.
And (3) sterilizing the dispersion liquid by high-pressure steam at 120 ℃ for 15 minutes, taking out after cooling to room temperature, and continuously freeze-drying for 24 hours to obtain the quercetin self-frame nano drug delivery system without the diselenide bond functional molecules, which is marked as HQu.
The self-framing nano-delivery system prepared in the above examples was subjected to structural and performance tests as follows.
The quercetin self-framing nano-delivery system prepared in example 1 was designated HQuSe. Quercetin, noted Qu. Hexachlorocyclotriphosphazene, noted HCCP. Selenocyamine, noted Se-Se. Mesalazine is 5-ASA. Dextran sodium sulfate is DSS. The quercetin nanometer drug delivery system without diselenide bond functional molecules is HQu.
Test results and analysis:
test 1: morphology, size and surface potential.
The quercetin prepared in the embodiment 1 is prepared from a frame nanometer drug delivery system (HQuSe) into a 50 mug/mL aqueous dispersion solution, and the aqueous dispersion solution is dripped on a copper mesh in a droplet form, and naturally dried for transmission electron microscope scanning test; the hydration diameter and surface potential were measured using a malvern particle sizer.
FIG. 1A is a transmission electron microscope image of a self-framing nano-drug delivery system of quercetin prepared in example 1; FIG. B is a transmission electron microscope image of the resveratrol self-framing nano-delivery system prepared in example 2; panel C is a transmission electron microscope image of the curcumin self-framing nano-delivery system prepared in example 3.
As can be seen from the A diagram in FIG. 1, the quercetin prepared in the example 1 is in the form of monodisperse nano microspheres from the frame nano drug delivery system. As can be seen from fig. 1B and C, the resveratrol self-framing nano-delivery system prepared in example 2 and the curcumin self-framing nano-delivery system prepared in example 3 are monodisperse nano-microspheres, have relatively uniform particle sizes, and have larger and more uniform particle sizes than the quercetin self-framing nano-delivery system prepared in example 1.
Figure 2 is a graph of the hydrated diameter of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of FIG. 2, the average hydrated diameter of quercetin self-framing nano-delivery system prepared in example 1 is 189.+ -. 6.8nm.
Figure 3 is a surface potential map of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of FIG. 3, the surface potential of quercetin prepared in example 1 was-50.4 mV from the surface of the framed nano drug delivery system.
Test 2: and (5) characterization of infrared spectrogram.
The quercetin prepared in example 1 was subjected to fourier transform infrared spectrometer testing from the framed nano drug delivery system for analysis of HQuSe component compositions.
FIG. 4 is an infrared spectrum of a quercetin self-framing nano-delivery system prepared in example 1. Wherein, the A graph is HQuSe infrared spectrogram; b is the infrared spectrum of Qu; panel C is the infrared spectrogram of HCCP; and D is the infrared spectrogram of Se-Se. HQuSe is a quercetin self-framing nano-delivery system prepared in example 1; qu is quercetin; HCCP is hexachlorocyclotriphosphazene; se-Se is selenocysteine.
As can be seen from the results of fig. 4, a new characteristic peak appears at 989cm -1 in panel a, which corresponds to the P-O-Ar bond of quercetin self-framed nano-delivery system; the characteristic peak intensities of P-Cl and P-N in hexachlorocyclotriphosphazene at 607cm -1 and 879cm -1 decrease. In addition, the characteristic peak of O-H bond of the quercetin in HQuSe at 3350cm -1 disappears, and in the explanation, the hydrogen atom of O-H bond in the quercetin is efficiently replaced by the chlorine atom of P-Cl bond in the hexachlorocyclotriphosphazene, which proves that the quercetin and the hexachlorocyclotriphosphazene monomer have nucleophilic substitution reaction. Notably, characteristic peaks of Se-C (740 cm -1) and Se-Se (545 cm -1) appear in HQuSe infrared spectra, confirming successful preparation of quercetin from the framed nano drug delivery system.
Test 3: and (5) evaluating the drug loading rate and the encapsulation rate.
The drug loading rate and encapsulation rate of quercetin prepared in example 1 from the frame nano drug delivery system (HQuSe) were measured by an ultraviolet-visible spectrometer according to the standard method specified in the guidelines of the 2010 edition (two parts of the Chinese pharmacopoeia). Fig. 5 is a graph of drug loading and encapsulation rate of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of fig. 5, the drug loading rate (DLE) and encapsulation rate (EE) of quercetin prepared in example 1 from the frame nano-delivery system were 68.9% and 49.8%, respectively, which are superior to most of the delivery systems reported in the literature.
Test 4: evaluation of Water dispersibility.
The water dispersibility of quercetin and quercetin prepared in example 1 from the framed nano drug delivery system was evaluated by visual direct observation, and recorded by photographing. Figure 6 is a graph of the water dispersibility of quercetin self-framing nano-delivery system prepared in example 1. Wherein Free Qu represents an aqueous dispersion of quercetin; HQuSe represents an aqueous dispersion of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of fig. 6, the quercetin prepared in example 1 was uniformly and stably dispersed in ultrapure water from the framed nano drug delivery system; however, the aqueous dispersion of quercetin showed severe coagulation, and the supernatant was cloudy.
Test 5: evaluation of physiological stability.
Evaluation of the dispersion stability of quercetin prepared in example 1 from the framed nano drug delivery system in ultrapure water, PBS buffer, serum (FBS) and cell culture medium (DMEM) by visual direct observation, photograph recording; the hydration diameters of these four physiological solutions were measured using a marvin particle sizer for 2 days of storage. FIG. 7 is a graph of the physiological stability of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of fig. 7, the quercetin prepared in example 1 was stably stored in ultrapure water, PBS buffer, serum (FBS) and cell culture medium (DMEM) for 2 days without the occurrence of a significant coagulation phenomenon, and accordingly, the hydrated diameter test did not have a significant dimensional change.
Test 6: evaluation of degradability.
The quercetin self-framing nano-delivery system (HQuSe) prepared in example 1 was tested for degradability in simulated gastric fluid, small intestinal fluid and colon fluid using transmission electron microscopy. Figure 8 is a graph of oral drug degradability of quercetin prepared in example 1 in a simulated gastrointestinal environment from a framed nano-drug delivery system.
As can be seen from the results of fig. 8, the quercetin self-framing nano-delivery system prepared in example 1 showed no significant morphological changes in gastric juice and small intestine simulated buffer; along with the extension of the co-incubation time, HQuSe generates nanometer microsphere expansion in the colorectal simulated buffer, the structure becomes loose, the spherical structure of HQuSe disappears after incubation for 24 hours, and the nanometer microsphere is degraded into ultra-small nanometer particles.
Test 7: evaluation of IBD treatment.
Animal model: using an IBD standard mouse animal model: c57BL/6J mice, females, 6 weeks old, weighing 18-20g, purchased from the university of Western An traffic laboratory animal center. Animals were kept under pathogen-free conditions: the ambient temperature is 25+/-2 ℃, and the relative constant humidity is 50+/-10%; can take water and food at will.
The acute colitis model is established by using Dextran Sodium Sulfate (DSS) (MP, USA) with molecular weight of 36000-5000. After 1 week of induction, the animals were randomly grouped into 6 groups of 8 animals each, as follows:
I-normal drinking water and PBS lavage were given as control group;
II-mice were given 3% DSS water and PBS lavage as negative controls;
III-mice were given 3% DSS drinking water and mesalamine (5-ASA) lavage as positive controls;
IV-mice were given 3% DSS drink and quercetin (Qu) lavage;
v-mice were given 3% DSS water and HQu lavage;
VI-mice were given 3% DSS water and HQuSe lavage.
Wherein, group I is marked as Control; group II is noted Colitis; group III is designated 5-ASA; group IV is denoted as Qu; group V is noted HQu; group VI is noted HQuSe.
The doses administered were normalized to 5-ASA (100 mg/kg/d) and Qu (100 mg/kg/d) and the system evaluated for the overall ROS levels, colon length, ROS content and permeability of colon tissue, and biosafety of IBD lesions. Figure 9 is a graph of ROS capture performance of quercetin self-framing nano-delivery systems prepared in example 1.
From the results of fig. 9, it can be seen that the quercetin self-framing nano-delivery system prepared in example 1 has high-efficiency elimination ability for ROS species indicators (DPPH, ABTS, and OH).
Figure 10 is a graph of the overall in vivo ROS levels and corresponding quantification of quercetin self-framing nano-delivery systems prepared in example 1. Wherein Total fluorescence represents Total fluorescence intensity.
As can be seen from the results of fig. 10, the quercetin self-frame nano-delivery system (HQuSe) prepared in example 1 significantly reduced the overall ROS levels at the acute colitis sites of the mice induced by Dextran Sodium Sulfate (DSS) and improved the performance by a factor of 3.4 compared to quercetin and the first-line clinical drug 5-ASA.
FIG. 11 is a graph of the therapeutic effect of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of fig. 11, the quercetin self-framing nano-delivery system (HQuSe) prepared in example 1 significantly eases and treats sodium dextran sulfate (DSS) -induced acute colitis in mice, maintains colon length, and protects colonic tissue integrity compared to quercetin and the clinical first-line drug 5-ASA.
FIG. 12 is a graph of the ROS level in colon tissue and colon permeability of a quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of FIG. 12, compared with quercetin and the first line clinical drug 5-ASA, quercetin prepared in example 1 had a ROS level strength of colon tissue as low as 0.78X10- 6 after treatment with the framed nano-delivery system (HQuSe), and colon permeability was improved by more than 2-fold.
Figure 13 is a safety profile of quercetin self-framing nano-delivery system prepared in example 1.
As can be seen from the results of FIG. 13, compared with quercetin and the first-line clinical drug 5-ASA, the quercetin prepared in example 1 was superior in biosafety from the framed nano drug delivery system (HQuSe), no obvious swelling of spleen occurred, and no index abnormality occurred in other important organ tissues.
From the results, the quercetin self-frame nano drug delivery system (HQuSe) prepared in the embodiment 1 of the invention is a monodisperse microsphere, the average size is 189+/-6.8 nm, the surface potential is-50.4 mV, and the drug loading rate (DLE) and the encapsulation rate (EE) are respectively as high as 68.9% and 49.8%. HQuSe has good water dispersion stability and physiological stability, and can be stored for a long time; HQuSe is capable of withstanding the extreme environment of the gastrointestinal tract while being effectively degraded in the inflammatory colonic environment. HQuSe negative surface charge and tissue adhesion, can achieve long-term enrichment at inflammatory colon sites, response and elimination of overloaded ROS achieve frame degradation and sustained release of natural polyphenolic compounds; can safely and effectively treat Inflammatory Bowel Disease (IBD).
Test 8: different natural polyphenols affect the structure and performance of the product.
TABLE 1 Structure and Property Effect of different Natural polyphenols on the products
From the results in table 1, it can be seen that three natural polyphenols, namely quercetin, resveratrol and curcumin, can undergo nucleophilic substitution reaction with functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers under the catalysis of acid binding agents, and are polymerized to form the self-framed nano drug delivery system. The three self-frame nanometer drug delivery systems are all monodisperse microspheres, have good water dispersion stability and physiological stability, and can be stored for a long time; can also resist the extreme environment of the gastrointestinal tract, and can be effectively degraded in inflammatory colon environment. The negative surface charge and tissue adhesiveness of the composition can realize long-term enrichment at inflammatory colon parts, and frame degradation and sustained release of natural polyphenol compounds can be realized by responding to and eliminating overloaded ROS; can safely and effectively treat inflammatory bowel disease; the ROS clearance rate is above 94%.
Test 9: the different dosage ratios of the functional molecules containing diselenide bond and hexachlorocyclotriphosphazene monomer affect the particle size and the dispersion performance of the product.
TABLE 2 influence of different dosage ratios of raw materials on particle size and dispersibility of the product
| Molar ratio of a | Particle size | Dispersity (PDI) | |
| Example 1 | 1:1 | 189±10.8nm | 0.107 |
| Example 4 | 2:1 | 119±5.5nm | 0.211 |
| Example 5 | 3:1 | 49±3.1nm | 0.396 |
Note that: the molar ratio a is the molar ratio of the functional molecule containing a diselenide bond to the hexachlorocyclotriphosphazene monomer. The smaller the dispersity (PDI) value, the more dispersed the product, and the better the effect.
As is clear from the results in Table 2, as the amount of the functional molecule containing a diselenide bond increases, the particle size of the obtained product gradually decreases, and the dispersion effect becomes worse. Comprehensive analysis shows that when the molar ratio of the functional molecules containing the diselenide bonds to the hexachlorocyclotriphosphazene monomer is 1-2:1, the dispersing effect of the product is relatively good, wherein when the molar ratio of the functional molecules containing the diselenide bonds to the hexachlorocyclotriphosphazene monomer is 1:1, the dispersing effect of the product is optimal.
Test 10: different functional molecules containing diselenide bonds have influence on the particle size and dispersion performance of the product.
TABLE 3 influence of different functional molecules containing diselenide bond on particle size and dispersibility of the product
Note that: the smaller the dispersity (PDI) value, the more dispersed the product, and the better the effect.
As shown in the results of Table 3, two different functional molecules containing diselenide bonds have certain influence on the particle size and the dispersion performance of the product, wherein the dispersion performance of the product prepared from selenocysteine is optimal.
Test 11: the dosage of the acid binding agent affects the particle size and the dispersion performance of the product.
TABLE 4 influence of the amount of acid-binding agent used on the particle size and dispersibility of the product
| Dosage of acid binding agent b | Particle size | Dispersity (PDI) | |
| Example 1 | 2％ | 189±10.8nm | 0.107 |
| Example 8 | 1％ | 234±13.1nm | 0.319 |
| Example 9 | 3％ | 392±20.6nm | 0.514 |
| Example 10 | 4％ | 1181±62.3nm | N/A |
| Example 11 | 5％ | 1431±35.8nm | N/A |
Note that: the dosage b of the acid binding agent is the volume percentage of the total volume of the mixed solution. The smaller the dispersity (PDI) value, the more dispersed the product, and the better the effect. N/A indicates undetected.
As can be seen from the results in Table 4, with the increase of the amount of the acid-binding agent, the particle size of the product tends to decrease and then increase, and when the amount of the acid-binding agent is 1-2%, the particle size of the prepared product is relatively small, and the dispersion effect is relatively good.
In conclusion, the hexa-functional cyclotriphosphazene monomer, the natural polyphenol containing polyphenol hydroxyl and the functional molecule containing diselenide bond are crosslinked and polymerized to prepare the oral nanometer medicament which has uniform particle size, biocompatibility, high stability, antioxidation, anti-inflammatory and better than the clinical first-line medicament aminosalicylic acid (5-ASA), solves the problems of poor structural stability, single treatment function, low medicament availability and the like of the traditional nanometer medicament delivery system, and enriches the medicament delivery system types for treating Inflammatory Bowel (IBD).
Wherein, natural polyphenol containing polyphenol hydroxyl, such as quercetin, etc., can improve the expression of Inducible Nitric Oxide Synthase (iNOS) involved in inflammation and immune response regulation by reducing the level of cytokines such as interferon (INF-gamma), tumor necrosis factor (tumor necrosis factor-alpha, TNF-alpha), interleukin (interleukin-1 beta, IL-1 beta), etc., thereby playing roles in inhibiting oxidative stress, anti-inflammation, reducing apoptosis, etc. on IBD, realizing the curative effects of relieving colonitis symptoms and maintaining the integrity of colon length and intestinal barrier. In addition, quercetin Pi Sufu contains phenolic hydroxyl groups, which imparts bioadhesive and antioxidant properties to HQuSe.
Selenocysteine containing diselenide bond is used as one of HQuSe effective components, can reduce target cell ROS level by directly clearing and up-regulating Nrf2/HO-1 signal path, exert antioxidant function, and finally realize IBD treatment and change intestinal ecology of IBD lack of selenium.
Hexachlorocyclotriphosphazene is taken as the center of HQuSe crosslinking structure, and active P-Cl bond can be subjected to nucleophilic substitution reaction with functional molecules containing polyphenol hydroxyl (e.g. phenolic hydroxyl of quercetin) and/or amino (e.g. amino of selenocysteine) under the catalysis of alkali, so that the hexachlorocyclotriphosphazene has good functionality and expansibility; meanwhile, the rich N-P six-membered ring skeleton and the highly cross-linked hybrid structure endow HQuSe with good structural stability; in addition, HQuSe has simple structural components, does not cause severe rejection and inflammatory reaction, and the degradation products are nontoxic small molecules such as phosphoric acid, ammonium salt, amino acid and the like, and can be normally metabolized out of the body. Therefore, based on the chemical characteristics of the open system and the customizable structure, the half-life period of the medicine can be prolonged, the bioavailability of the functional medicines such as quercetin and the like can be improved, the micro-environment responsive degradation of IBD inflammation and the sustained release of the medicine can be realized, and toxic byproducts are not generated.
In summary, the self-framed nano drug delivery system obtained by the invention can effectively resist the extreme environment of the gastrointestinal tract by oral administration, and the effect that the colon part is negatively charged HQuSe interacts with positively charged inflamed colon tissues through electrostatic adsorption, hydrogen bond, cation-pi interaction and covalent bond is achieved. Meanwhile, the phenolic hydroxyl group of the quercetin in the HQuSe self-frame nano drug delivery system further endows HQuSe with active oxygen scavenging property and tissue adhesiveness, and prolongs the residence time of the quercetin in the colon. HQuSe is very sensitive to endocytosis by macrophages due to long term enrichment and cell affinity of quercetin. The intracellular response and the elimination of ROS through enzymatic degradation and selenocysteine further control the release of quercetin to synergistically enhance the anti-inflammatory and antioxidant effects, and finally realize the high-efficiency treatment of IBD.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method of preparing a self-framing nano-drug delivery system, comprising the steps of:
dissolving natural polyphenol, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers in a solvent to obtain a mixed solution; wherein the molar ratio of the sum of the amount of the natural polyphenol substance and the amount of the diselenide bond-containing functional molecule substance to the hexachlorocyclotriphosphazene monomer is 1-3:1;
mixing the mixed solution with an acid binding agent, and reacting under the catalysis of the acid binding agent to prepare the self-frame nano drug delivery system.
2. The method for preparing a self-framing nano-drug delivery system according to claim 1, wherein the natural polyphenol is any one of quercetin, resveratrol and curcumin.
3. The method of claim 1, wherein the functional molecule containing diselenide is any one of selenocyamine and 4,4' -diselenidenediyldianiline.
4. The method of claim 1, wherein the molar ratio of natural polyphenols to functional molecules containing diselenide linkages is 1:1-2.
5. The method for preparing a self-framing nano drug delivery system according to claim 1, wherein the acid binding agent is any one of triethylamine and sodium hydroxide solution.
6. The method of claim 5, wherein the molar concentration of the sodium hydroxide solution is between 0.05 and 0.125mol/L.
7. The method of claim 1, wherein the amount of acid binding agent is 1-5% of the total volume of the mixed solution.
8. The method of claim 1, wherein the solvent is acetonitrile or tetrahydrofuran.
9. A self-framing nano-delivery system prepared by the method of any one of claims 1-8.
10. Use of a self-framing nano-delivery system for the manufacture of a medicament for the treatment of inflammatory bowel disease, wherein the self-framing nano-delivery system is as defined in claim 9.
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2024
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