CN113520992A - Ligustrazine nanocrystal, ligustrazine nanocrystal temperature-sensitive hydrogel, preparation method and application thereof - Google Patents
Ligustrazine nanocrystal, ligustrazine nanocrystal temperature-sensitive hydrogel, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a ligustrazine nanocrystal, a ligustrazine nanocrystal temperature-sensitive hydrogel, a preparation method and application thereof, and relates to the technical field of medicines. The ligustrazine thermosensitive hydrogel is composed of ligustrazine nanocrystals, poloxamer and a solubilizer vitamin E polyethylene glycol succinate, and the traditional Chinese medicine monomer ligustrazine is wrapped in the nanocrystal micelles. The eye temperature sensitive hydrogel is prepared from temperature sensitive hydrogel based on poloxamer copolymer, and carries insoluble drug ligustrazine. The ligustrazine temperature-sensitive hydrogel has a temperature-dependent physical crosslinking mode capable of rapidly generating sol-gel conversion, has a good slow release effect, small drug particle size, strong tissue penetration, increased drug solubility, improved drug loading rate, an encapsulation rate of more than 95 percent, high bioavailability and biocompatibility and no obvious systemic toxic and side effects. The preparation method has the advantages of simple process, short time consumption, low cost and convenient popularization. The invention has good clinical application prospect and development value.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a ligustrazine nanocrystal, a ligustrazine nanocrystal temperature-sensitive hydrogel, a preparation method and application thereof.
Background
Retinal Neovascularization (RNV) and Choroidal Neovascularization (CNV) are associated with a variety of ocular diseases, such as Diabetic Retinopathy (DR), wet age-related macular degeneration (nAMD), retinopathy of prematurity, and central and branch Retinal vein occlusion. In developed countries, nAMD is the leading cause of blindness in people over 50 years of age, characterized by the formation of CNVs. CNV formation, which results from immature neovasculature disrupting bruch's membrane and growing from the underlying choroid to the outer retina, is a process involving inflammation and angiogenesis.
In the treatment methods for fundus hemorrhagic disease, the Anti-neovascular drug therapy of intravitreal injection of Anti-vascular endothelial growth factor inhibitor (Anti-VEGF) has been considered as the first-line treatment strategy for CNV, and although these drugs have achieved certain curative effect in treating neovascular eye diseases, they have great limitations at the same time. The main disadvantages are that the medicine has larger molecular weight and shorter curative effect, and the clearance rate of vitreous protein is high, so that repeated injection is needed in the vitreous cavity of eyes, and the occurrence probability of complications such as infection, bleeding, vascular embolism and the like is greatly increased. Not all patients benefit from this treatment. Some patients need repeated injections and even treatment for the whole life, and the treatment effect is not ideal.
Rhizoma Ligustici Chuanxiong is used as traditional Chinese herbal medicine, and bioactive component 2, 3, 5, 6 Tetramethylpyrazine (TMP), also called ligustrazine, has effects of promoting blood circulation for removing blood stasis, resisting oxidation, regulating blood lipid, and improving hemorheology, and is commonly used for treating coronary heart disease and angina pectoris. The underlying therapeutic mechanisms of ligustrazine include modulation of the immune system and enhancement of anti-inflammatory effects. Ligustrazine is a fat-soluble drug, has low solubility in water, and how to increase the solubility in an aqueous solvent to facilitate absorption is a challenge in drug development.
CNV requires a sustained release of the active substance to achieve successful efficacy, and the development of new active drugs with sustained and effective release avoids the rapid clearance of the drug and reduces the risk of multiple injections. The temperature-sensitive hydrogel material is a low-viscosity polymer aqueous solution at the environmental temperature, and the hydrogel can improve the drug entrapment performance through the solubilization of micelles, thereby realizing the long-acting slow release of the drug, achieving good biocompatibility and safe and effective release of the drug in a target organ, serving as a drug warehouse and prolonging the residence time of the drug in eyes. The drug carrier based on the nano composite temperature-sensitive hydrogel releases the drug from the nano crystal particles to the hydrogel and then diffuses from the hydrogel to the eyes, so that the drug carrier has a better slow release effect.
Disclosure of Invention
The first purpose of the invention is to provide the ligustrazine nanocrystal temperature-sensitive hydrogel which has high drug loading and encapsulation rate of more than 95%, can be used for long-acting slow release in vitreous bodies, and reduces the injection frequency.
The second objective of the present invention is to provide a ligustrazine nanocrystal which can be used for ocular injection.
The third purpose of the invention is to provide a preparation method of the ligustrazine nanocrystal, which has simple process and uniform crystal grain size and can be used for preparing the subsequent temperature-sensitive hydrogel.
The fourth purpose of the invention is to provide a preparation method of the ligustrazine nanocrystal temperature-sensitive hydrogel, which has the advantages of simple process, short time consumption, low production cost, safety and no toxicity in the whole process, and convenience in popularization.
The fifth purpose of the invention is to provide an application of the ligustrazine nanocrystal temperature-sensitive hydrogel in preparing a temperature-sensitive hydrogel nano drug delivery system for treating ocular hemorrhagic diseases.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
In a first aspect, an embodiment of the present application provides a ligustrazine nanocrystal temperature-sensitive hydrogel, in which ligustrazine nanocrystals are wrapped in the temperature-sensitive hydrogel, and the ligustrazine nanocrystal temperature-sensitive hydrogel is used for ocular injection.
In a second aspect, the present application provides a ligustrazine nanocrystal prepared from ligustrazine and poloxamer.
In a third aspect, an embodiment of the present application provides a preparation method of the ligustrazine nanocrystal, including the following steps of dissolving ligustrazine and poloxamer in an organic solvent, removing the organic solvent containing the drug by rotary evaporation and vacuum drying, hydrating, rotating, and performing an ice bath ultrasonic method to obtain a ligustrazine nanocrystal preparation.
In a fourth aspect, the present application provides a preparation method of a ligustrazine nanocrystal temperature-sensitive hydrogel, which is to add poloxamer and a solubilizer into the ligustrazine nanocrystal preparation, stir in an ice bath and mix uniformly to obtain the ligustrazine nanocrystal temperature-sensitive hydrogel.
In a fifth aspect, an embodiment of the present application provides an application of the aforementioned ligustrazine nanocrystal temperature-sensitive hydrogel in preparing a temperature-sensitive hydrogel nano drug delivery system for treating ocular hemorrhagic diseases.
Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:
first, ligustrazine is a fat-soluble and insoluble drug, and how to increase the solubility of ligustrazine in an aqueous solution is a challenge in drug development. CNV requires a sustained release of the active substance to achieve a successful therapeutic effect, and it is energetically efficient and imperative to develop new formulations that can sustain an effective release of the active drug, which can also avoid rapid clearance of the drug and reduce the risk of multiple injections. The invention combines the advantage that the temperature-sensitive hydrogel generates reversible sol-gel change based on the change of environmental temperature, designs the ligustrazine nanocrystal temperature-sensitive hydrogel with improved solubility and penetrating power, and can realize in-situ slow release effect on local focuses, thereby exerting the local long-term application curative effect of the traditional Chinese medicine monomer.
Second, the duration of action of a drug administered intravitreally may depend in part on the length of time the injected drug remains at the site of administration. The longer the intravitreal half-life, the longer the duration of the therapeutic response is expected. The temperature-sensitive hydrogel material is a low-viscosity polymer aqueous solution at the ambient temperature, and can be easily used for drug encapsulation and intravitreal injection administration. When the administration environment exceeds the critical gelation temperature, the physical crosslinking mode of rapid gelation can be changed, so that local in-situ sustained-release administration is realized, the release of the loaded drug is effectively prolonged with effective biocompatibility and safety, and the drug storage is formed to prolong the residence time of the drug in eyes. Therefore, according to the ligustrazine nanocrystal temperature-sensitive hydrogel provided by the invention, the ligustrazine nanocrystals are encapsulated by the temperature-sensitive hydrogel, so that the drug can be targeted on the primary focus, the toxic and side effects of the whole body are reduced, and meanwhile, the hydrogel can play a local long-acting slow-release effect and has a long-time treatment effect on symptoms. The preparation can reduce the injection frequency, reduce the dependence of patients on the medicine, stabilize the blood concentration at the treatment concentration for a long time, and play a sustained-release role, thereby improving the curative effect of the medicine and increasing the compliance of the patients.
The preparation method of the ligustrazine nanocrystal temperature-sensitive hydrogel provided by the invention has the advantages of simple process, short time consumption, low production cost, easiness in industrial production and large-scale popularization, and good industrial prospect. Meanwhile, the preparation method provided by the invention has the advantages of high drug loading and better treatment effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the particle size of ligustrazine nanocrystals (TMP-NCs) in example 1 of the present invention;
FIG. 2 is a particle size diagram of a temperature-sensitive hydrogel (TMP-NCs-gel) of ligustrazine nanocrystals in example 1 of the present invention.
FIG. 3 is a transmission electron micrograph (TEM image) of ligustrazine nanocrystals (TMP-NCs) in example 2 of the present invention.
FIG. 4 is a transmission electron micrograph (TEM image) of a tetramethylpyrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) in example 2 of the present invention;
FIG. 5 is a graph showing the particle size of ligustrazine nanocrystals (TMP-NCs) in example 2 of the present invention;
FIG. 6 is a particle size diagram of a temperature-sensitive hydrogel (TMP-NCs-gel) of ligustrazine nanocrystals in example 2 of the present invention;
FIG. 7 is the in vitro sustained release curve of ligustrazine nanocrystals (TMP-NCs) and ligustrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) in example 2 of the present invention;
FIG. 8 is a graph showing the particle size of ligustrazine nanocrystals (TMP-NCs) in example 3 of the present invention;
FIG. 9 is a particle size diagram of a temperature-sensitive hydrogel (TMP-NCs-gel) of ligustrazine nanocrystals in example 3 of the present invention;
FIG. 10 is a graph showing the particle size of ligustrazine nanocrystals (TMP-NCs) in example 4 of the present invention;
FIG. 11 is a particle size diagram of a temperature-sensitive hydrogel (TMP-NCs-gel) of ligustrazine nanocrystals in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
In a first aspect, an embodiment of the present application provides a ligustrazine nanocrystal temperature-sensitive hydrogel, in which ligustrazine nanocrystals are wrapped in the temperature-sensitive hydrogel, and the ligustrazine nanocrystal temperature-sensitive hydrogel is used for ocular injection.
In some embodiments of the present invention, in the above ligustrazine nanocrystal temperature-sensitive hydrogel, the phase transition temperature of the ligustrazine nanocrystal temperature-sensitive hydrogel is 24 ℃ to 25 ℃, the temperature-sensitive hydrogel is selected from poloxamer block copolymer hydrogel, the mass concentration of the poloxamer in the ligustrazine nanocrystal temperature-sensitive hydrogel is 15% to 25%, and the poloxamer block copolymer hydrogel is used for loading ligustrazine nanocrystals.
In some embodiments of the invention, in the above ligustrazine nanocrystal temperature-sensitive hydrogel, the ligustrazine nanocrystal is an aqueous solvent prepared from a traditional Chinese medicine monomer ligustrazine and poloxamer, and the mass ratio of the ligustrazine to the poloxamer in the aqueous solvent is 1: 2.5-1: 4.
In some embodiments of the present invention, the ligustrazine nanocrystal temperature-sensitive hydrogel further includes vitamin E polyethylene glycol succinate, and the concentration of the vitamin E polyethylene glycol succinate is 0.05-0.2% (w/w).
In some embodiments of the present invention, in the above ligustrazine nanocrystal temperature-sensitive hydrogel, the concentration of ligustrazine in the ligustrazine nanocrystal temperature-sensitive hydrogel is 0.5mg/ml to 4 mg/ml.
In some embodiments of the present invention, in the above ligustrazine nanocrystal temperature-sensitive hydrogel, the concentration of ligustrazine in the ligustrazine nanocrystal temperature-sensitive hydrogel is 0.5mg/ml to 2.75 mg/ml.
In some embodiments of the present invention, the particle size of the ligustrazine nanocrystal and the ligustrazine nanocrystal temperature-sensitive hydrogel is 100-1000 nm; the particle size of the ligustrazine nanocrystal temperature-sensitive hydrogel is 10 nm-1000 nm.
In some embodiments of the present invention, the particle size of the ligustrazine nanocrystal and the ligustrazine nanocrystal temperature-sensitive hydrogel is 100-200 nm; the particle size of the ligustrazine nanocrystal temperature-sensitive hydrogel is 10-200 nm.
In a second aspect, the present application provides a ligustrazine nanocrystal prepared from ligustrazine and poloxamer.
In a third aspect, an embodiment of the present application provides a preparation method of the ligustrazine nanocrystal, including the following steps of dissolving ligustrazine and poloxamer in an organic solvent, removing the organic solvent in which the drug is dissolved by rotary evaporation and vacuum drying, hydrating, rotating, and performing ice bath ultrasound to obtain a ligustrazine nanocrystal preparation.
In some embodiments of the present invention, the preparation method of the ligustrazine nanocrystal comprises the specific steps of drying a mixture of organic solvents in which ligustrazine and poloxamer are dissolved in vacuum for 30-90 min, removing all the organic solvents until the drugs are separated out to form a drug membrane, adding distilled water with the same amount as the initial organic solvent into the drug membrane to dissolve the drug membrane into the distilled water, hydrating for 30-60 min, then vortexing for 5-15 min, and ultrasonically crushing in ice bath of 20-40W for 10-30 min to obtain the ligustrazine nanocrystal preparation.
In a fourth aspect, the present application provides a preparation method of a ligustrazine nanocrystal temperature-sensitive hydrogel, which is to add poloxamer and a solubilizer into the ligustrazine nanocrystal preparation, stir in an ice bath and mix uniformly to obtain the ligustrazine nanocrystal temperature-sensitive hydrogel.
In a fifth aspect, an embodiment of the present application provides an application of the aforementioned ligustrazine nanocrystal temperature-sensitive hydrogel in preparing a temperature-sensitive hydrogel nano drug delivery system for treating ocular hemorrhagic diseases.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present embodiment aims to provide a ligustrazine nanocrystal and a ligustrazine nanocrystal temperature-sensitive hydrogel, which are mainly prepared through the following steps:
firstly, ligustrazine and poloxamer F127 as a non-ionic surfactant are respectively weighed and placed in an eggplant-shaped bottle according to the weight ratio of 1:2.5, an organic solvent dichloromethane with the corresponding concentration and the equivalent dose is added, then evaporating dichloromethane on a rotary evaporator, setting the temperature of the rotary evaporator to be 40 ℃ of the boiling point of the dichloromethane, vacuum drying for 40 min with vacuum drying pump and drying oven, vacuum-pumping to remove organic solvent dichloromethane, vacuum-pumping dichloromethane containing ligustrazine and poloxamer to form medicinal membrane at the bottom of the bottle, adding distilled water equal to the initial amount of organic solvent into the bottle to dissolve the membrane at the bottom of the bottle in distilled water, then hydrating on a rotary evaporator for 30 minutes, then vortexing on a vortexing instrument for 5 minutes, and finally carrying out 20W ultrasound on the cells in an ultrasonic cell crushing instrument for 25 minutes by an ice bath method to obtain the ligustrazine nanocrystal preparation.
The ligustrazine nanocrystal temperature-sensitive hydrogel is prepared by the following ice bath method, specifically comprising the following steps: surfactant F127 and solubilizer D-alpha-vitamin E polyethylene glycol succinate are added into the ligustrazine nanocrystal preparation, so that the final mass concentration of the surfactant is 15% (w/w) and the final mass concentration of the solubilizer is 0.1% (w/w). The mixture was then stirred in an ice bath with a magnetic stirrer until the surfactant and solubilizer were completely dissolved, resulting in a clear and clear solution (TMP-NCs-Gel) and samples were stored as a solid Gel at Room Temperature (RT). In the gel, the concentration of ligustrazine is 0.5 mg/ml.
Particle size and potential difference measurements were performed on ligustrazine nanocrystals (TMP-NCs) and ligustrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) by a Malvern Zetasizer (ZEN3600 Nano ZS, Worcestershire, UK) particle size analyzer, respectively, fig. 1 is a particle size diagram of ligustrazine nanocrystals (TMP-NCs), the particle size of which is 213.4nm, the dispersion coefficient Pdi of which is 0.876, and the potential difference of which is-5.05; FIG. 2 is a particle size diagram of tetramethylpyrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel), the particle size is 191nm, the dispersion coefficient Pdi is 0.667, the potential difference is-5.71, and the encapsulation efficiency is more than 95%.
Example 2
The purpose of the present embodiment is to provide a ligustrazine nanocrystal and a ligustrazine nanocrystal temperature-sensitive hydrogel, which is mainly prepared through the following steps:
firstly, ligustrazine and poloxamer F127 as a non-ionic surfactant are respectively weighed and placed in an eggplant-shaped bottle according to the weight ratio of 1:3, an organic solvent dichloromethane with the corresponding concentration and the equivalent dose is added, then evaporating dichloromethane on a rotary evaporator, setting the temperature of the rotary evaporator to be 37 ℃ of the boiling point of the dichloromethane, vacuum-drying for 60min under vacuum drying pump and drying oven, vacuum-pumping to remove organic solvent dichloromethane, vacuum-pumping to obtain medicinal film with ligustrazine and poloxamer dissolved in dichloromethane at the bottom of the bottle, adding distilled water equal to the initial amount of organic solvent into the bottle, dissolving the film at the bottom of the bottle in distilled water, and then hydrating on a rotary evaporator for 40 minutes, then vortexing on a vortexing instrument for 10 minutes, and finally performing 30W ultrasonic treatment on the cells in an ultrasonic cell crushing instrument for 15 minutes by an ice bath method to obtain the ligustrazine nanocrystal preparation.
The ligustrazine nanocrystal temperature-sensitive hydrogel is prepared by the following ice bath method, specifically comprising the following steps: surfactant F127 and solubilizer D-alpha-vitamin E polyethylene glycol succinate are added into the ligustrazine nanocrystal preparation, so that the final mass concentration of the surfactant is 20% (w/w) and the final mass concentration of the solubilizer is 0.1% (w/w). The mixture was then stirred in an ice bath with a magnetic stirrer until the surfactant and solubilizer were completely dissolved, resulting in a clear and clear solution (TMP-NCs-Gel) and samples were stored as a solid Gel at Room Temperature (RT). In the gel, the concentration of ligustrazine is 1 mg/ml.
Particle sizes of the ligustrazine nanocrystals (TMP-NCs) and the ligustrazine nanocrystal temperature-sensitive hydrogel (TMP-NCs-gel) are observed by an electron microscope, and the result is shown in FIG. 3, wherein the particle size is uniform as shown in FIG. 3, the four images in FIG. 3 are transmission electron microscope images (different magnifications) of the ligustrazine nanocrystals (TMP-NCs), and the ligustrazine nanocrystals (TMP-NCs) are rod-shaped structures as shown in the figure. The four images in fig. 4 are transmission electron microscope images (different magnifications) of tetramethylpyrazine nanocrystal temperature-sensitive hydrogel (TMP-NCs-gel), and when the nanocrystals are made into gel, the form of the nanocrystals changes to form a non-flowing transparent solid, and the temperature-sensitive hydrogel with a microscopic form is spherical nanoparticles.
Particle size and potential difference measurements were performed on ligustrazine nanocrystals (TMP-NCs) and ligustrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) by a Malvern Zetasizer (ZEN3600 Nano ZS, Worcestershire, UK) particle size analyzer, respectively, fig. 5 is a particle size diagram of ligustrazine nanocrystals (TMP-NCs), the particle size of which is 149.7nm, the dispersion coefficient Pdi of which is 1.0, and the potential difference of which is-5.23; FIG. 6 is a particle size diagram of tetramethylpyrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel), the particle size is 132.8nm and 10.35nm, the dispersion coefficient Pdi is 0.309, the potential difference is-9.52, and the encapsulation efficiency is greater than 95%. Fig. 7 is an in vitro sustained release curve of 1mg/ml ligustrazine nanocrystal and ligustrazine temperature sensitive hydrogel (TMP-NCs-gel), and as can be seen from fig. 7, the ligustrazine nanocrystal temperature sensitive hydrogel prepared in this embodiment can achieve a slow and stable release effect compared to ligustrazine nanocrystal.
Example 3
The purpose of the present embodiment is to provide a ligustrazine nanocrystal and a ligustrazine nanocrystal temperature-sensitive hydrogel, which is mainly prepared through the following steps:
firstly, ligustrazine and a non-ionic surfactant poloxamer F127 are respectively weighed and placed in an eggplant-shaped bottle according to the weight ratio of 1:3.5, an organic solvent dichloromethane with equivalent concentration is added, then dichloromethane is rotationally evaporated on a rotary evaporator, the temperature of the rotary evaporator is set to be 38 ℃ of the boiling point of dichloromethane, meanwhile, a vacuum drying pump and a drying oven are used for vacuum-pumping drying for 70 minutes, the organic solvent dichloromethane is removed by vacuum-pumping, a medicine film formed after the dichloromethane organic solvent in which the ligustrazine and the poloxamer are dissolved is visible at the bottom of the eggplant-shaped bottle after vacuum-pumping, distilled water with equivalent amount to the initial amount of the organic solvent is added in the eggplant-shaped bottle, the film at the bottom of the eggplant-shaped bottle is dissolved in the distilled water, then the film is hydrated on the rotary evaporator for 50 minutes, then the film is vortexed on a vortexing instrument for 15 minutes, and finally, 40W ultrasound is carried out for 25 minutes in an ultrasonic cell crushing instrument by an ice bath method, to obtain the ligustrazine nanocrystal preparation.
The ligustrazine nanocrystal temperature-sensitive hydrogel is prepared by the following ice bath method, specifically comprising the following steps: poloxamer F127 and solubilizer D-alpha-vitamin E polyethylene glycol succinate are added into the ligustrazine nanocrystal preparation, so that the final concentration of the F127 is 25% (w/w) and the final concentration of the solubilizer is 0.15% (w/w). The mixture was then stirred in an ice bath with a magnetic stirrer until the poloxamer and the solubilizing agent were completely dissolved, resulting in a clear solution (TMP-NCs-Gel) and the sample was stored as a solid Gel at Room Temperature (RT). In the gel, the concentration of ligustrazine is 2.75 mg/ml.
Particle size and potential difference measurements were performed on ligustrazine nanocrystals (TMP-NCs) and ligustrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) by a Malvern Zetasizer (ZEN3600 Nano ZS, Worcestershire, UK) particle size analyzer, respectively, fig. 8 is a particle size diagram of ligustrazine nanocrystals (TMP-NCs), the particle size was 312.6nm, the dispersion coefficient Pdi was 0.621, and the potential difference was-7.65; FIG. 9 is a particle size diagram of a tetramethylpyrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel), the particle sizes are 9.002nm and 112.9nm, the dispersion coefficient Pdi is 0.329, the potential difference is-14.6, and the encapsulation efficiency is more than 95%.
Example 4
The purpose of the present embodiment is to provide a ligustrazine nanocrystal and a ligustrazine nanocrystal temperature-sensitive hydrogel, which is mainly prepared through the following steps:
firstly, ligustrazine and a non-ionic surfactant poloxamer F127 are respectively weighed and placed in an eggplant-shaped bottle according to the weight ratio of 1:4, an organic solvent dichloromethane with equivalent concentration is added, then dichloromethane is rotationally evaporated on a rotary evaporator, the temperature of the rotary evaporator is set to be 40 ℃ of the boiling point of dichloromethane, meanwhile, a vacuum drying pump and a drying box are used for vacuum drying for 90 minutes, the organic solvent dichloromethane is removed by vacuum suction, a medicine film formed by vacuum pumping of the dichloromethane organic solvent dissolved with the ligustrazine and the poloxamer can be seen at the bottom of the eggplant-shaped bottle, distilled water with equivalent amount to the initial amount of the organic solvent is added in the eggplant-shaped bottle, the film at the bottom of the eggplant-shaped bottle is dissolved in the distilled water, then the film is hydrated on the rotary evaporator for 60 minutes, then the film is vortexed on a vortexing instrument for 15 minutes, and finally, 40W ultrasound is carried out in an ultrasonic cell crushing instrument for 30 minutes by an ice bath method, to obtain the ligustrazine nanocrystal preparation.
The ligustrazine nanocrystal temperature-sensitive hydrogel is prepared by the following ice bath method, specifically comprising the following steps: surfactant F127 and solubilizer D-alpha-vitamin E polyethylene glycol succinate are added into the ligustrazine nanocrystal preparation, so that the final concentration of F127 is 25% (w/w) and the final concentration of the solubilizer is 0.15% (w/w). The mixture was then stirred in an ice bath with a magnetic stirrer until the poloxamer and the solubilizing agent were completely dissolved, resulting in a clear solution (TMP-NCs-Gel) and the sample was stored as a solid Gel at Room Temperature (RT). In the gel, the concentration of ligustrazine is 4 mg/ml.
Particle size and potential difference measurements were performed on ligustrazine nanocrystals (TMP-NCs) and ligustrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel) by a Malvern Zetasizer (ZEN3600 Nano ZS, Worcestershire, UK) particle size analyzer, respectively, fig. 9 is a particle size diagram of ligustrazine nanocrystals (TMP-NCs), the particle size was 818.9nm, the dispersion coefficient Pdi was 0.289, and the potential difference was-12.1; FIG. 11 is a particle size diagram of a tetramethylpyrazine nanocrystal temperature sensitive hydrogel (TMP-NCs-gel), the particle sizes include 6.839nm, 16.27nm and 254.7nm, the dispersion coefficient Pdi is 0.447, the potential difference is-13.3, and the encapsulation efficiency is greater than 95%.
In addition to the use of F127 in the foregoing examples, other types of poloxamers may be used.
In summary, the ligustrazine nanocrystal temperature-sensitive hydrogel provided by the embodiment of the invention has the following advantages:
first, ligustrazine is a fat-soluble and insoluble drug, and how to prepare the ligustrazine into a water-soluble preparation is a challenge of drug development. CNV requires a sustained release of the active substance to achieve successful therapeutic results, and the development of new formulations capable of sustained and effective release of the active drug is of great significance for the clinical treatment of CNV, which also avoids the rapid clearance of the drug and reduces the risk of multiple injections. The preparation takes poloxamer temperature-sensitive hydrogel as a carrier to entrap ligustrazine nanocrystals, can achieve the optimal in-situ long-acting slow-release effect, enables the blood concentration to be stable at the treatment concentration for a long time, and plays the roles of slow release and local effect to the greatest extent, thereby improving the curative effect of the medicine, increasing the compliance of patients, and having better treatment effect compared with other temperature-sensitive hydrogel carriers.
Second, the duration of action of a drug administered intravitreally may depend in part on the length of time the injected drug remains at the site of administration. The longer the intravitreal half-life, the longer the duration of the therapeutic response is expected. The disadvantages of intravitreal injection can be alleviated by formulation modifications and optimization of the pharmaceutical dosage form. The phase transition temperature of the ligustrazine nanocrystal temperature-sensitive hydrogel provided by the invention is 24-25 ℃, and under the environment in the vitreous body of eyes, nanocrystal micelles gradually expand to form a thick gel barrier on the surface to directly reach focuses, so that the ligustrazine nanocrystal temperature-sensitive hydrogel has a long-time treatment effect on symptoms, and has no obvious other adverse reactions of the whole body. Meanwhile, the preparation can reduce the injection times and the dependence of patients on the medicine, and can stabilize the blood concentration at the treatment concentration for a long time to play the roles of slow release and local effect, thereby improving the curative effect of the medicine and increasing the compliance of the patients.
The preparation method of the ligustrazine temperature-sensitive hydrogel provided by the invention has the advantages of simple process, short time consumption, low production cost, easiness in industrial production and large-scale popularization, and good industrial prospect. Meanwhile, the preparation is directly injected to the posterior segment of the eye by a relatively noninvasive local vitreous cavity injection method, the preparation is quickly converted into a transparent semi-solid gel storage at body temperature, the local slow release therapeutic effect is achieved, the medicine safety is high, and both the ligustrazine crystal preparation and the ligustrazine nanocrystal temperature-sensitive hydrogel preparation have transparent properties. The eyeball is a refraction imaging visual light conversion organ with high light transmission, and is an important sense organ for exchanging information and emotion between human and the external environment. Good vision has high requirements on an eye refractive imaging system, so the poloxamer temperature-sensitive hydrogel is expected to play an important role in the field of eyes with higher transparency requirements, particularly fundus diseases. The invention has good clinical application prospect and development value.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A ligustrazine nanocrystal temperature-sensitive hydrogel is characterized in that ligustrazine nanocrystals are wrapped in the temperature-sensitive hydrogel, and the ligustrazine nanocrystal temperature-sensitive hydrogel is used for eye injection.
2. The ligustrazine nanocrystal temperature-sensitive hydrogel according to claim 1, wherein the phase transition temperature of the ligustrazine nanocrystal temperature-sensitive hydrogel is 24-25 ℃, the temperature-sensitive hydrogel is selected from poloxamer block copolymer hydrogel, the mass concentration of the poloxamer block copolymer hydrogel in the ligustrazine nanocrystal temperature-sensitive hydrogel is 15-25%, and the poloxamer block copolymer hydrogel is used for loading ligustrazine nanocrystals.
3. The ligustrazine nanocrystal temperature-sensitive hydrogel according to claim 1, wherein the ligustrazine nanocrystal is an aqueous solvent prepared from a traditional Chinese medicine monomer ligustrazine and poloxamer, and the mass ratio of the ligustrazine to the poloxamer in the aqueous solvent is 1: 2.5-1: 4.
4. The ligustrazine nanocrystal temperature-sensitive hydrogel according to claim 1, wherein the concentration of ligustrazine in the ligustrazine nanocrystal temperature-sensitive hydrogel is 0.5 mg/ml-4 mg/ml.
5. The ligustrazine nanocrystal temperature-sensitive hydrogel according to claim 1, wherein the particle size of the ligustrazine nanocrystal is 100-1000 nm; the particle size of the ligustrazine nanocrystal temperature-sensitive hydrogel is 10 nm-1000 nm.
6. A ligustrazine nanocrystal is characterized by being prepared from ligustrazine and poloxamer.
7. A process for preparing the nano-class Chuangxionzine crystal as claimed in claim 6, which comprises dissolving Chuangxionzine and poloxamer in organic solvent, rotary evaporating, vacuum drying, hydrating, and ultrasonic treating in ice bath.
8. The preparation method of ligustrazine nanocrystals, according to claim 7, wherein the drying comprises vacuum drying the mixture of organic solvents in which ligustrazine and poloxamer are dissolved for 30-90 min, removing all organic solvents until the drugs are precipitated to form a drug film, adding distilled water with the same amount as the initial organic solvent into the drug film to dissolve the drug film in distilled water, hydrating for 30-60 min, then vortexing for 5-15 min, and ultrasonically crushing in 20-40W ice bath for 10-30 min to obtain the ligustrazine nanocrystal preparation.
9. A preparation method of ligustrazine nanocrystal temperature-sensitive hydrogel, which is characterized in that poloxamer and solubilizer are added into the ligustrazine nanocrystal preparation prepared in claim 7 or 8, and the mixture is stirred and mixed evenly in ice bath to obtain the ligustrazine nanocrystal temperature-sensitive hydrogel.
10. The use of a ligustrazine nanocrystal temperature sensitive hydrogel according to any one of claims 1 to 5 in the preparation of a temperature sensitive hydrogel nano drug delivery system for the treatment of ocular hemorrhagic diseases.
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