CN113750039A - Baicalin-calcium alginate temperature-sensitive nanogel, preparation method and application thereof - Google Patents
Baicalin-calcium alginate temperature-sensitive nanogel, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of baicalin-calcium alginate temperature-sensitive nanogel. The preparation method comprises the following steps: (1) mixing sodium alginate solution (containing baicalin) and oil phase, mixing with calcium chloride ethanol solution, stirring, and separating water phase to obtain baicalin-calcium alginate nanometer gel suspension; (2) and (3) uniformly mixing and dissolving 6 parts by weight of baicalin-calcium alginate nano gel, 1 part by weight of poloxamer 188 and 7 parts by weight of poloxamer 407, and preparing the baicalin-calcium alginate temperature-sensitive nano gel. The invention constructs the baicalin-calcium alginate temperature-sensitive nano gel, adopts a nasal administration mode, leads the baicalin to bypass a blood brain barrier and quickly reach the brain after being absorbed by nasal mucosa, and quickly plays a role in treating acute stroke. Compared with the traditional oral and intravenous preparations for treating cerebral apoplexy, the invention has the advantages of quick effect, small dosage, convenient use and the like.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicines, in particular to a preparation method and application of baicalin-calcium alginate temperature-sensitive nanogel for treating cerebral apoplexy.
Background
The 2017 Chinese disease burden research result published in the 2019 Lancet magazine shows that stroke is the first cause of death of people in China. Chinese reports of stroke prevention and treatment (2018) also indicate that, at present, people in 40 years old and older in China have stroke in 1242 thousands, and 196 thousands of people die of stroke each year. Its high morbidity, mortality and disability rate place a heavy burden on society and families. The prevention and treatment of stroke in China still face huge challenges, and the prevention and treatment system needs to be further strengthened urgently.
Tissue Plasminogen Activator (tPA) is a unique drug approved by the FDA in the United states for treating acute ischemic stroke, and due to the side effects of short treatment time window, high cerebral hemorrhage transformation risk and the like, the tPA is limited to be widely applied in clinic, practically beneficial groups are few in clinical use, and only 3% -5% of patients with ischemic stroke can obtain tPA venous thrombolytic therapy. How to better exert the effect of the medicine on the treatment of the acute stroke has important significance on the rescue of patients with the acute stroke.
Disclosure of Invention
The invention aims to provide baicalin-calcium alginate temperature-sensitive nanogel, a preparation method and application thereof aiming at the defects of the prior art. The baicalin-calcium alginate-loaded temperature-sensitive nanogel constructed by the invention is liquid at normal temperature, and a nasal administration mode is adopted, so that the liquid at the normal temperature is changed into gel to be adhered to the surface of a nasal cavity by the temperature of the nasal cavity, and the baicalin is absorbed by nasal cavity mucosa and then rapidly reaches the brain by bypassing the blood brain barrier, thereby rapidly playing the role of treating acute stroke. Compared with the traditional oral and intravenous preparations for treating cerebral apoplexy, the invention has the advantages of quick effect, small dosage, convenient use and the like.
The technical scheme adopted by the invention is as follows:
in a first aspect, a method for preparing a baicalin-calcium alginate temperature-sensitive nanogel is provided, wherein the baicalin-calcium alginate temperature-sensitive nanogel is prepared by adding pharmaceutically effective auxiliary materials into bulk drug baicalin, and specifically comprises the following steps:
(1) preparation of baicalin-calcium alginate nanogel
According to 500-200: 5-1.5: 1, respectively adding liquid paraffin, Span 80 and Tween 80, and fully dispersing in water bath at 40-60 ℃ and 500rpm to obtain an oil phase. Adjusting the stirring rotation speed to 1000rpm, dropping 60-300 parts of sodium alginate solution containing baicalin into the oil phase, controlling the flow rate to be 0.75mL/min, continuously stirring after dropping is finished, fully and uniformly mixing the water phase and the oil phase, adding 20-100 parts of curing agent, controlling the flow rate to be 0.25mL/min, stirring at the rotation speed of 1000rpm, continuously stirring for 1h after dropping is finished, centrifuging the obtained nanogel at 3000rpm for 15min, separating the water phase to obtain baicalin-calcium alginate nanogel suspension, quickly freezing by using liquid nitrogen, and freeze-drying. Wherein, in the sodium alginate solution containing baicalin, the concentration of sodium alginate is 1-10mg/mL, the concentration of baicalin is 0.1-20mg/mL, and the curing agent is a 70% ethanol solution of calcium chloride of 0.5-5 mg/mL.
(2) Preparation of baicalin-calcium alginate temperature-sensitive nano gel
And (2) taking 6 parts by weight of the baicalin-calcium alginate nanogel prepared in the step (1), adding normal saline, uniformly dispersing, then adding 1 part by weight of poloxamer 188 (P188) and 7 parts by weight of poloxamer 407 (P407) in an ice bath, and mixing until the materials are completely dissolved uniformly. Preparing the baicalin-calcium alginate temperature-sensitive nanogel.
Further, the baicalin-calcium alginate temperature-sensitive nano gel is in a gel shape at the temperature of 30 +/-2 ℃.
Further, the weight ratio of the liquid paraffin, the Span 80 and the Tween 80 is 333.33 parts by weight: 2.33 parts by weight: 1 part by weight; the weight portion of the sodium alginate solution containing the baicalin is 100 weight portions, and the weight portion of the curing agent is 33.33 weight portions.
In a second aspect, a baicalin-calcium alginate temperature-sensitive nanogel obtained by the preparation method of the first aspect.
In a third aspect, the baicalin-calcium alginate temperature-sensitive nanogel in the second aspect is used for preparing a medicine for treating cerebral apoplexy, and the medicine is administrated through a nasal cavity.
In some embodiments, the stroke comprises ischemic stroke and hemorrhagic stroke.
In a fourth aspect, a medicine for treating cerebral apoplexy comprises an effective amount of the baicalin-calcium alginate temperature-sensitive nanogel of the first aspect.
The invention has the beneficial effects that: the baicalin-calcium alginate temperature-sensitive nano gel has the effect of treating acute stroke, and has the advantages of definite curative effect, high safety and controllable quality; the temperature-sensitive nanogel constructed by the invention is liquid at normal temperature, and adopts a nasal administration mode, the temperature of the nasal cavity enables the liquid at the normal temperature to be changed into gel to be adhered to the surface of the nasal cavity, so that the baicalin can bypass the blood brain barrier to quickly reach the brain after being absorbed by the nasal cavity mucosa, and the effect of treating acute stroke can be quickly exerted. Compared with the traditional oral and intravenous preparations for treating cerebral apoplexy, the invention has the advantages of quick effect, small dosage, convenient use and the like. The preparation method is simple, the process conditions are mild, the manufacturing cost is low, and the pharmaceutical composition has the advantages of pharmacogenomics for treating the stroke and is suitable for clinical application and market popularization.
Drawings
The invention is further explained below with reference to the figures and examples;
FIG. 1 is TTC picture of rat brain tissue of model group, baicalin (vein) and baicalin-calcium alginate temperature sensitive nano gel group.
Fig. 2 shows the statistical results of the cerebral infarction volumes of rats in the model group, baicalin (vein) and baicalin-calcium alginate temperature-sensitive nanogel group, wherein P is less than 0.05.
Detailed Description
The invention provides baicalin-calcium alginate temperature-sensitive nano gel for treating cerebral apoplexy, which is prepared from baicalin as a main material, and a calcium alginate temperature-sensitive nano gel system comprising sodium alginate, calcium chloride, poloxamer 188 and poloxamer 407.
The preparation method comprises the following steps:
(1) preparation of baicalin-calcium alginate nanogel
According to 500-200: 5-1.5: 1, respectively adding liquid paraffin, Span 80 and Tween 80, and fully dispersing in water bath at 40-60 ℃ and 500rpm to obtain an oil phase. Adjusting the stirring rotation speed to 1000rpm, dropping 60-300 parts of sodium alginate solution containing baicalin into the oil phase, controlling the flow rate to be 0.75mL/min, continuously stirring after dropping is finished, fully and uniformly mixing the water phase and the oil phase, adding 20-100 parts of curing agent, controlling the flow rate to be 0.25mL/min, stirring at the rotation speed of 1000rpm, continuously stirring for 1h after dropping is finished, centrifuging the obtained nanogel at 3000rpm for 15min, separating the water phase to obtain baicalin-calcium alginate nanogel suspension, quickly freezing by using liquid nitrogen, and freeze-drying. Wherein, in the sodium alginate solution containing baicalin, the concentration of sodium alginate is 1-10mg/mL, the concentration of baicalin is 0.1-20mg/mL, and the curing agent is a 70% ethanol solution of calcium chloride of 0.5-5 mg/mL.
(2) Preparation of baicalin-calcium alginate temperature-sensitive nano gel
And (2) putting 6 parts by weight of the baicalin-calcium alginate nanogel prepared in the step (1) into a proper penicillin bottle, paving the nanogel at the bottom of the penicillin bottle, adding a small amount of physiological saline, and electromagnetically stirring until the nanogel is uniformly dispersed. Adding a proper amount of crushed ice into a large beaker, placing a penicillin bottle in the middle for precooling, and adjusting the rotating speed and the position of the penicillin bottle to ensure that the rotor is uniformly stirred. Then 1 part by weight of poloxamer 188 (P188) and 7 parts by weight of poloxamer 407 (P407) are mixed according to the prescription amount, and the mixture is added little by little until the components are completely dissolved uniformly. Storing the prepared baicalin-calcium alginate temperature-sensitive nano gel in a refrigerator at 4 ℃.
The calcium alginate nano gel is prepared by a conventional preparation method according to the above proportion, and the following components are 333.33 parts by weight of liquid paraffin, Span 80 and Tween 80: 2.33 parts by weight: 1 part by weight; the purpose and effect of the present invention will become more apparent by describing the present invention in detail by taking the example of the part by weight of the sodium alginate solution containing baicalin as 100 parts by weight and the part by weight of the solidifying agent as 33.33 parts by weight.
Example 1:
1. preparation of baicalin-calcium alginate gel
150mL of liquid paraffin, 1.05mL of Span 80 and 0.45mL of Tween 80 were added to a 500mL three-necked flask, and thoroughly dispersed in a water bath at 40 ℃ and 500rpm for 30min to serve as an oil phase. Adjusting the stirring speed to 1000rpm, slowly dripping 45mL of a sodium alginate solution (containing 27mg of baicalin, and prepared into a 5mg/mL sodium alginate solution containing 0.6mg/mL of baicalin) with the concentration of 5mg/mL into the oil phase by using a peristaltic pump, controlling the flow rate to be 0.75mL/min, and continuously stirring for 1h after finishing dripping to fully and uniformly mix the water phase and the oil phase. Adding 15mL of 70% ethanol solution of calcium chloride (1mg/mL) as curing agent by using a peristaltic pump, controlling the flow rate to be 0.25mL/min, stirring at the rotation speed of 1000rpm, continuously stirring for 1h after dropwise adding is finished, pouring the obtained nano gel into a 50mL centrifuge tube, weighing, centrifuging at 3000rpm for 15min, separating a water phase to obtain baicalin-calcium alginate gel suspension, quickly freezing by using liquid nitrogen, freezing, drying and storing for later use.
2. Preparation of baicalin-calcium alginate temperature-sensitive nano gel
Chopping the baicalin-calcium alginate nanogel by a sterilized knife, accurately weighing 360mg of the baicalin-calcium alginate nanogel, placing the obtained product into a penicillin bottle (2.7cm multiplied by 4.7cm), paving the nanogel at the bottom of the bottle (no need to hang the wall), adding 2.16ml of physiological saline, adding a rotor, and electromagnetically stirring until the nanogel is uniformly dispersed (about 30 min). Adding a proper amount of crushed ice into a 500ml beaker, placing a penicillin bottle in the middle for precooling, taking care that the ice does not cover a bottle cap, and adjusting the rotating speed and the position of the penicillin bottle to ensure that the rotor is uniformly stirred (a glass culture dish can be buckled on the stirrer in an inverted mode, and then the beaker is placed on the stirrer, so that the rotor can rotate stably). Then 60mg of poloxamer 188 (P188) and 420mg of poloxamer 407 (P407) are mixed according to the prescription amount, and the mixture is added little by little (added in 15 times) by using weighing paper until the mixture is completely dissolved uniformly, and the mixture is taken care not to be added to the bottle wall after about 1 hour. And subpackaging the prepared temperature-sensitive nanogel into 200uL centrifuge tubes, packaging by using tinfoil paper and storing in a refrigerator at 4 ℃.
The baicalin-calcium alginate temperature-sensitive nano gel can not flow when the test tube is turned over within the range of 30 +/-2 ℃ as determined by simulating the gelation condition of the gel in the nasal cavity by a test tube tilting method and a viscometer. The gel with the viscosity range can achieve the effect of delaying the medicine from flowing out of the nasal cavity after nasal administration, and simultaneously ensures that the medicine can be rapidly released from a medium and rapidly enters the brain through the nasal cavity to treat diseases.
Example 2:
pharmacodynamic evaluation experiment on cerebral ischemia reperfusion injury by using baicalin-calcium alginate temperature-sensitive nano gel to administer through nasal cavity
1. Laboratory animal
Healthy Wistar rats, male, weight 260-.
2. Experimental methods
2.1 rat cerebral ischemia model preparation and evaluation
A rat Middle Cerebral Artery Occlusion (MCAO) model is established by adopting a line embolism method, and cortical cerebral blood flow is monitored by a Laser Doppler flow meter (LDF) to evaluate whether molding is successful or not, specifically: after rats were anesthetized with isoflurane gas, a skull was drilled 2mm behind bregma and 5mm lateral to the right side of midline to form a thin hole, and an LDF fiber probe was fixed thereto to continuously monitor cerebral regional blood flow (CBF) in the cerebral cortex. And monitoring before molding to obtain a baseline value, and continuously monitoring until the postoperative blood flow value is stable. The rat is in a supine position, the rat is disinfected conventionally, the skin is prepared, muscle fascia is stripped, the right Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are separated and exposed in sequence, the branches of the ECA are separated, the ECA is ligated in a double-line mode, the CCA and the ICA are clamped close to the proximal heart end, a V-shaped incision is obliquely cut at the proximal heart end of the ECA, a thread plug selected according to the weight of the rat is inserted rapidly, and the silk thread which is placed at the root of the ECA in advance is tied into a single knot. The ECA is detached between the double nodes, the arterial clamp on the ICA is removed, the ECA is gently pulled into approximate alignment with the ICA, and a wire plug is slowly inserted cranially toward the ICA until some resistance is felt to block blood flow in the lateral Middle Cerebral Artery (MCA). The LDF showed that MCA dominated the cortical local blood flow to be reduced to less than 25% of the blood flow basal value at this time, and the modeling was successful. After 2h of occlusion the plug was slowly withdrawn and the ECA severed end was ligated, the CCA artery clamp was released and the skin was sutured. The heating plate for the small animals maintains the anal temperature of 37 ℃ in the rat operation, the temperature is kept at 30 ℃ after the operation, 5mL of physiological saline is injected into the abdominal cavity to prevent dehydration, and the rat is put back into the cage after waking up and freely takes food and water.
2.2 Experimental groups and administrations
The experimental animals were randomly divided into 3 groups of 10 rats each, and the groups were as follows:
(1) model group: after 10min of cerebral ischemia, 150. mu.L/kg of physiological saline was nasally administered to both sides, respectively.
(2) Baicalin intravenous administration group: baicalin solution (15mg/kg) was injected into tail vein after 10min of cerebral ischemia.
(3) Baicalin-calcium alginate temperature-sensitive nano-gel group: after cerebral ischemia is carried out for 10min, baicalin-calcium alginate temperature-sensitive nano gel (0.4mg/kg is calculated according to baicalin to be 167.7 mu L/kg) is administrated to both noses every time, and in order to avoid rat asphyxiation caused by simultaneous administration of two sides, the medicine is administrated to the infarct side nasal cavity for 10min and then the medicine is administrated to the normal side nasal cavity. The gel was allowed to solidify in the nasal cavity by continued anesthesia for 10min after administration to both nostrils.
The total dose was 3 times, 10 minutes, 24 hours and 48 hours after cerebral ischemia. The rats were sacrificed 72h after cerebral ischemia and brains were removed.
2.3 cerebral infarction volume evaluation
After 72 hours of cerebral ischemia, rats are anesthetized with isoflurane gas, and perfused with heart normal saline, brains are taken and placed in a brain slice mold (removing olfactory bulbs and cerebellum) to sequentially pass through 2 mm-thick coronal slices from front to back, and are stained for 15min in 2% TTC solution (TTC is a fat-soluble photosensitive compound, a mitochondrial respiratory chain proton receptor, dehydrogenase in normal brain tissue reacts with TTC to form red, and ischemic brain tissue cannot react with TTC due to the reduction of the activity of the mitochondrial dehydrogenase of cell membranes and forms a white dye-repellent area), and are analyzed by using Image J Image analysis software. The specific formula is as follows:
cerebral infarction volume (%) - (normal lateral hemisphere volume-normal partial volume of ischemic lateral hemisphere) ÷ normal lateral hemisphere volume × 100%.
3. Results of the experiment
3.1 Effect of baicalin-calcium alginate temperature-sensitive nanometer gel on cerebral infarction volume of rat in acute cerebral ischemia phase
After the brain tissue of the rat is dyed by TTC, the normal brain tissue is uniformly red-dyed, and white dye-repellent areas (infarcted foci) can be seen in the areas of striatum, hippocampus, cortex and the like of the ischemic hemisphere brain tissue of the model group, thereby indicating that the model is successfully made; compared with the model group, the nasal administration (0.4mg/kg) and the intravenous administration (15mg/kg) of the baicalin-calcium alginate temperature-sensitive nano gel can reduce the cerebral infarction volume after cerebral apoplexy, and the nasal administration (0.4mg/kg) dose of the baicalin-calcium alginate temperature-sensitive nano gel is only 1/37.5 of the intravenous injection baicalin dose (15mg/kg) (figure 1 and figure 2).
The baicalin-calcium alginate temperature-sensitive nano gel is proved to have obvious protective effect on the acute cerebral apoplexy injury by nasal administration. The nasal administration dosage (0.4mg/kg) of the baicalin-calcium alginate temperature-sensitive nano gel is only 1/37.5 of the intravenous injection baicalin dosage (15mg/kg), and the administration dosage is small and the curative effect is better.
The invention constructs the baicalin-calcium alginate-loaded temperature-sensitive nano gel, the normal temperature state is liquid, the nasal cavity administration mode is adopted, the temperature of the nasal cavity enables the liquid in the normal temperature state to be changed into gel to be adhered to the surface of the nasal cavity, the baicalin can bypass the blood brain barrier to quickly reach the brain after being absorbed by the nasal cavity mucosa, the effect of treating acute cerebral apoplexy is quickly exerted, the medicine dosage is reduced, and the generation of corresponding side effects is also reduced. Compared with the traditional oral and intravenous preparations for treating cerebral apoplexy, the invention has the advantages of quick effect, small dosage, convenient use and the like.
The above embodiments are intended to illustrate rather than to limit the invention, and any modifications and variations of the present invention are within the spirit of the invention and the scope of the claims.
Claims (5)
1. A preparation method of baicalin-calcium alginate temperature-sensitive nanogel is characterized in that the baicalin-calcium alginate temperature-sensitive nanogel is prepared by adding pharmaceutically effective auxiliary materials into bulk drug baicalin, and specifically comprises the following steps:
(1) preparation of baicalin-calcium alginate nanogel
According to 500-200: 5-1.5: 1, respectively adding liquid paraffin, Span 80 and Tween 80, and fully dispersing in water bath at 40-60 ℃ and 500rpm to obtain an oil phase. Adjusting the stirring rotation speed to 1000rpm, dropping 60-300 parts of sodium alginate solution containing baicalin into the oil phase, controlling the flow rate to be 0.75mL/min, continuously stirring after dropping is finished, fully and uniformly mixing the water phase and the oil phase, adding 20-100 parts of curing agent, controlling the flow rate to be 0.25mL/min, stirring at the rotation speed of 1000rpm, continuously stirring for 1h after dropping is finished, centrifuging the obtained nanogel at 3000rpm for 15min, separating the water phase to obtain baicalin-calcium alginate nanogel suspension, quickly freezing by using liquid nitrogen, and freeze-drying. Wherein, in the sodium alginate solution containing baicalin, the concentration of sodium alginate is 1-10mg/mL, the concentration of baicalin is 0.1-20mg/mL, and the curing agent is a 70% ethanol solution of calcium chloride of 0.5-5 mg/mL.
(2) Preparation of baicalin-calcium alginate temperature-sensitive nano gel
And (2) taking 6 parts by weight of the baicalin-calcium alginate nanogel prepared in the step (1), adding normal saline, uniformly dispersing, then adding 1 part by weight of poloxamer 188 (P188) and 7 parts by weight of poloxamer 407 (P407) in an ice bath, and mixing until the materials are completely dissolved uniformly. Preparing the baicalin-calcium alginate temperature-sensitive nanogel.
2. The preparation method according to claim 1, wherein the baicalin-calcium alginate temperature-sensitive nanogel is gel-like at a temperature of 30 ± 2 ℃.
3. The preparation method according to claim 1, wherein the weight ratio of the liquid paraffin, the Span 80 and the Tween 80 is 333.33 parts by weight: 2.33 parts by weight: 1 part by weight; the weight portion of the sodium alginate solution containing the baicalin is 100 weight portions, and the weight portion of the curing agent is 33.33 weight portions.
4. A baicalin-calcium alginate temperature-sensitive nanogel prepared by the preparation method of any one of claims 1-3.
5. An application of the baicalin-calcium alginate temperature-sensitive nanogel of claim 4 in preparing a medicine for treating cerebral apoplexy.
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