NL2033322B1

NL2033322B1 - Baicalin-calcium alginate thermosensitive nanogel as well as preparation method and application thereof

Info

Publication number: NL2033322B1
Application number: NL2033322A
Authority: NL
Inventors: Zhang Shanshan; Fan Xiang; Zhou Yuan; Chen Zhong
Original assignee: Univ Zhejiang Chinese Medical
Priority date: 2021-10-26
Filing date: 2022-10-14
Publication date: 2023-06-21
Also published as: CN113750039B; CN113750039A; NL2033322A

Abstract

Disclosed are a preparation method and application of baicalin— calcium alginate thermosensitive nanogel. The preparation method includes the following steps: (1) mixing a sodium alginate solution (containing baicalin) with an oil phase fully, stirring the resulting solution with a calcium chloride ethanol solution evenly, and separating a water phase to obtain a baicalin—calcium alginate nanogel suspension; and (2) mixing 6 parts by weight of baicalin—calcium alginate nanogel, 1 part by weight of poloxamer 188 and 7 parts by weight of poloxamer 407 and dissolving the mixture evenly to obtain baicalin—calcium alginate thermosensitive nanogel. The baicalin—calcium alginate thermosensitive nanogel is administered through nasal cavity, so that the baicalin is absorbed, by the nasal mucosa and quickly reaches the brain. by bypassing the blood brain barrier. Compared with traditional oral and intravenous preparations for treating stroke, the present invention has the advantages of rapid, onset, small dosage and convenient use.

Description

BAICALIN-CALCIUM ALGINATE THERMOSENSITIVE NANOGEL AS WELL AS

PREPARATION METHOD AND APPLICATION THEREOF

TECHNICAL FIELD

The application relates to the technical field of traditional

Chinese medicine, and in particular to a preparation method and application of baicalin-calcium alginate thermosensitive nanogel for treating stroke.

BACKGROUND ART

The Study on Global Burden of Disease 2017 in China published by the Lancet in 2019 shows that stroke has become the leading cause of death in China. The Brief Report on stroke Prevention and

Treatment in China (2018) also points out that there are 12.42 million stroke cases in Chinese residents aged 40 and above at present, and 1.96 million patient die from stroke every year. The high incidence, high mortality and high disability rate put a heavy burden on society and families. The stroke prevention and treatment in China still faces great challenges, and the preven- tion and treatment system needs to be further strengthened.

The tissue Plasminogen Activator (tPA) is the only drug ap- proved by FDA for treating acute ischemic stroke. However, tPA's wide clinical application is limited due to side effects such as short treatment time window and high risk of cerebral hemorrhage transformation. In clinical application, the actual benefit popu- lation is very small, only 3%-5% of patients with ischemic stroke can receive intravenous thrombolytic therapy with tPA. Therefore, how to better play the effect of drugs in the treatment of acute stroke is of great significance for the rescue of patients with acute stroke.

SUMMARY

In order to overcome the deficiencies in the prior art, the present invention is intended to provide baicalin-calcium alginate thermosensitive nanogel as well as a preparation method and an ap-

plication thereof. The baicalin-calcium alginate thermosensitive nanogel prepared by the present invention is liquid at room tem- perature and administered through nasal cavity, wherein the tem- perature of nasal cavity makes the liquid at room temperature be- come gelatinous and adhere to the surface of nasal cavity, so that the baicalin is absorbed by the nasal mucosa and quickly reaches the brain by bypassing the blood-brain barrier, so as to play a role of treating acute stroke quickly. Compared with traditional oral and intravenous preparations for treating stroke, the present invention has the advantages of rapid onset, small dosage and con- venient use.

The technical solutions of the present invention are specifi- cally as follows:

The first aspect provides a preparation method of baicalin- calcium alginate thermosensitive nanogel, wherein the baicalin- calcium alginate thermosensitive nanogel is prepared from an ac- tive pharmaceutical ingredient baicalin and pharmaceutically ef- fective auxiliary materials, specifically including the following steps: (1) preparation of baicalin-calcium alginate nanogel adding liquid paraffin, Span 80 and Tween 80 respectively at a weight ratio of 500-200:5-1.5:1, and fully dispersing the mix- ture in a water bath at 40-60°C and 500 rpm as an oil phase; ad-

Justing a stirring speed to 1,000 rpm, and dropping 60-300 parts of sodium alginate solution containing baicalin into the oil phase at a flow rate of 0.75 mL/min; upon completion of dropping, stir- ring the mixed solution continuously until the water phase is mixed with the oil phase thoroughly; adding 20-100 parts of curing agent at a flow rate of 0.25 mL/min under stirring at a speed of 1,000 rpm; upon completion of dropping, stirring the mixture con- tinuously for 1 h, centrifuging the resulting nanogel at 3,000 rpm for 15 min, separating the water phase to obtain a baicalin- calcium alginate nanogel suspension, and quick-freezing with lig- uid nitrogen and freeze-drying the baicalin-calcium alginate nano- gel suspension; wherein in the sodium alginate solution containing baicalin, a sodium alginate concentration is 1-10 mg/mL, a bai- calin concentration is 0.1-20 mg/mL, and the curing agent is a 70%

ethanol solution of 0.5-5 mg/mL calcium chloride. (2) preparation of baicalin-calcium alginate thermosensitive nanogel adding normal saline to 6 parts by weight of baicalin-calcium alginate nanogel prepared in S1, dispersing the nanogel evenly, adding 1 part by weight of poloxamer 188 (P 188) and 7 parts by weight of poloxamer 407 (P 407) under an ice bath condition, and mixing the mixture until all the materials are dissolved evenly to obtain baicalin-calcium alginate thermosensitive nanogel.

Further, the baicalin-calcium alginate thermosensitive nano- gel is gelatinous at 30+2°C.

Further, a weight ratio of the liquid paraffin to the Span 80 to the Tween 80 is 333.33:2.33:1; the amount of the sodium algi- nate solution containing baicalin is 100 parts by weight, and the amount of the curing agent is 33.33 parts by weight.

The second aspect provides baicalin-calcium alginate thermo- sensitive nanogel prepared by the preparation method in the first aspect.

The third aspect provides an application of the baicalin- calcium alginate thermosensitive nanogel in the second aspect in preparing a stroke treatment drug, wherein the drug is adminis- tered through nasal cavity.

In some embodiments, the stroke includes ischemic stroke and hemorrhagic stroke.

The fourth aspect provides a stroke treatment drug, including an effective amount of the baicalin-calcium alginate thermosensi- tive nanogel in the first aspect.

The present invention has the following beneficial effects:

The baicalin-calcium alginate thermosensitive nanogel of the pre- sent invention has the effect of treating acute stroke with defi- nite efficacy, high safety and controllable quality. The thermo- sensitive nanogel prepared by the present invention is liquid at room temperature and administered through nasal cavity, wherein the temperature of nasal cavity makes the liquid at room tempera- ture become gelatinous and adhere to the surface of nasal cavity, so that the baicalin is absorbed by the nasal mucosa and quickly reaches the brain by bypassing the blood-brain barrier, so as to play a role of treating acute stroke quickly. Compared with tradi- tional oral and intravenous preparations for treating stroke, the present invention has the advantages of rapid onset, small dosage and convenient use. Moreover, the preparation method is simple, the technological conditions are mild, and the manufacturing cost is low. Thus, the baicalin-calcium alginate thermosensitive nano- gel has pharmacoeconomic advantages for stroke treatment, and is suitable for clinical application and marketing.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below in com- bination with the accompanying drawings and embodiments.

FIG. 1 is TTC pictures of the brain tissues of rats in a mod- el group, a baicalin (intravenous) group and a baicalin-calcium alginate thermosensitive nanogel group.

FIG. 2 shows statistical results of the cerebral infarction volume of rats in the model group, the baicalin (intravenous) group and the baicalin-calcium alginate thermosensitive nanogel group (*P < 0.05).

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides baicalin-calcium alginate thermosensitive nanogel for treating stroke, wherein a main mate- rial is baicalin, and a calcium alginate thermosensitive nanogel system includes sodium alginate, calcium chloride, poloxamer 188 and poloxamer 407.

A preparation method of the present invention includes the following steps: {1) Preparation of baicalin-calcium alginate nanogel liquid paraffin, Span 80 and Tween 80 are added respectively at a weight ratio of 500-200:5-1.5:1, and fully dispersed in a wa- ter bath at 40-60°C and 500 rpm as an oil phase; a stirring speed is adjusted to 1,000 rpm, and 60-300 parts of sodium alginate so- lution containing baicalin is dropped into the oil phase at a flow rate of 0.75 mL/min; upon completion of dropping, the mixed solu- tion is stirred continucusly until the water phase is mixed with the oil phase thoroughly; 20-100 parts of curing agent is added at a flow rate of 0.25 mL/min under stirring at a speed of 1,000 rpm; upon completion of dropping, the mixture is stirred continuously for 1 h, the resulting nanogel is centrifuged at 3,000 rpm for 15 min, the water phase is separated to obtain a baicalin-calcium al- 5 ginate nanogel suspension, and the baicalin-calcium alginate nano- gel suspension is quick-frozen with liquid nitrogen and freeze- dried; wherein in the sodium alginate solution containing bai- calin, a sodium alginate concentration is 1-10 mg/mL, a baicalin concentration is 0.1-20 mg/mL, and the curing agent is a 70% etha- nol solution of 0.5-5 mg/mL calcium chloride. {2) Preparation of baicalin-calcium alginate thermosensitive nanogel 6 parts by weight of baicalin-calcium alginate nanogel pre- pared in S1 is placed in a suitable vial, and the nanogel is spread on a bottom of the vial, a small amount of normal saline is added, and the mixture is electromagnetically stirred until the nanogel is dispersed evenly; an appropriate amount of crushed ice is added to a beaker, and the vial is put in the middle of the beaker for precooling, a speed and a position of the vial are ad- justed so that a rotor stirs the mixture evenly; 1 part by weight of poloxamer 188 (P 188) and 7 parts by weight of poloxamer 407 (P 407) are mixed according to a prescription dosage, and the result- ing mixture is added little by little until all the materials are dissolved evenly; the prepared baicalin-calcium alginate thermo- sensitive nanogel is stored in a refrigerator at 4°C.

Wherein the calcium alginate nancgel can be prepared at the above-mentioned ratios by a conventional preparation method. The present invention will be described in detail below with an exam- ple as follows: a weight ratio of the liquid paraffin to the Span 80 to the Tween 80 is 333.33:2.33:1; the amount of the sodium al- ginate solution containing baicalin is 100 parts by weight, and the amount of the curing agent is 33.33 parts by weight. Thus, the purpose and effect of the present invention will become more obvi- ous.

Example 1: 1. Preparation of baicalin-calcium alginate gel 150 mL of liquid paraffin, 1.05 mL of Span 80 and 0.45 mL of

Tween 80 were added to a 500 mL three-necked bottle respectively, and fully dispersed in a water bath at 40°C and 500 rpm for 30 min as an oil phase. A stirring speed was adjusted to 1,000 rpm, and 45 mL of 5 mg/mL sodium alginate solution (containing 27 mg of baicalin, dissolved to a clear solution ultrasonically in advance, and prepared into a 5 mg/mL sodium alginate solution containing 0.6 mg/mL baicalin) was slowly dropped into the oil phase by a peristaltic pump at a flow rate of 0.75 mL/min; upon completion of dropping, the mixed solution was stirred continuously for 1 h un- til the water phase was mixed with the oil phase thoroughly. 15 mL of 70% ethanol solution of curing agent (1 mg/mL) calcium chloride was added by the peristaltic pump at a flow rate of 0.25 mL/min and a stirring speed of 1,000 rpm; upon completion of dropping, the resulting solution was stirred continuously for 1 h, and the resulting nanogel was poured into a 50 mL centrifuge tube and weighed, and centrifuged at 3,000 rpm for 15 min, the water phase was separated to obtain a baicalin-calcium alginate gel suspen- sion, and the baicalin-calcium alginate gel suspension was quick- frozen with liquid nitrogen and freeze-dried for storage and later use. 2. Preparation of baicalin-calcium alginate thermosensitive nanogel

The baicalin-calcium alginate nanogel was cut up with a ster- ilized knife, 360 mg of baicalin-calcium alginate nanogel was ac- curately weighed and placed in a vial (2.7 cm x 4.7 cm), and the nanogel was spread on a bottom of the vial (not to hang on the wall). 2.16 ml of normal saline was added, and a rotor was placed, the mixture was electromagnetically stirred until the nanogel was dispersed evenly (about 30 min). An appropriate amount of crushed ice was added to a 500 ml beaker, and the vial was put in the mid- dle of the beaker for precooling (the ice should not be beyond the vial cap), and a speed and a position of the vial were adjusted so that the rotor stirred the mixture evenly (a glass culture dish could be inverted on the agitator, and the beaker was placed on the glass culture dish, so that the rotor could work stably). 60 mg of poloxamer 188 (P 188) and 420 mg of poloxamer 407 (P 407) were mixed according to a prescription dosage, and the resulting mixture was added (15 times) little by little with weighing paper until all the materials were dissolved evenly, with a time of about 1 h (the mixture should not be added onto the vial wall).

The prepared thermosensitive nanogel was sub-packaged to a 200 ul centrifuge tube, packaged with tin foil paper and stored in a re- frigerator at 4°C.

The baicalin-calcium alginate thermosensitive nanogel of the present invention may remain static when the tube is inverted within the range of 30+2°C by simulating gelation of the gel in the nasal cavity through a tube tilt method and determination by vis- cometer. The gel within such viscosity range can achieve the ef- fect of delaying the outflow of the drug from the nasal cavity af- ter nasal administration, and also ensure that the drug can be quickly released from the medium and quickly enter the brain through the nasal cavity to treat the disease.

Example 2:

Pharmacodynamic evaluation experiment on cerebral ischemia- reperfusion injury by the baicalin-calcium alginate thermosensi- tive nanogel of the present invention through nasal administration 1. Experimental animal

Healthy Wistar rats, male, weighing 260-280 g, provided by

Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were fasted for 12 h before the experiment, but were allowed to drink water freely. 2. Experimental method 2.1 Preparation and evaluation of a cerebral ischemia model in rats

A middle cerebral artery occlusion (MCAC) model in rats was established by a suture method, and the cortical cerebral blood flow was monitored by Laser Doppler flowmetry (LDF) to evaluate the success of model, specifically as follows: after the rats were anesthetized with isoflurane, a thin hole was drilled in the skull 2 mm posterior to the bregma and 5 mm adjacent to the right side of the midline, and a LDF fibre-optics probe was fixed here to monitor the cerebral blood flow (CBF) in the cerebral cortex con- tinuously. A baseline value was obtained by monitoring before mod- eling and continucusly monitored until a postoperative blood flow value was stable. In supine position, after routine disinfection, skin preparation and muscle fascia dissection of the rats, the common carotid artery (CCA), external carotid artery (ECA) and in- ternal carotid artery (ICA) were successively separated and ex- posed, the ECA’s branch was transected, the ECA was ligatured by a double wire, the proximal parts of CCA and ICA were occluded, a “V-shaped incision was cut on the bias at the proximal part of

ECA and the suture selected according to the rat’s body weight was inserted quickly, and a single knot was tied to the silk thread pre-placed at the root of the ECA. The ECA was transected between the double knot, the artery clamp was removed from the ICA, the

ECA was gently pulled to form an approximate straight line with the ICA, and the suture was inserted slowly into the into-cranial direction of the ICA until a little resistance was felt to ob- struct the blood flow of the middle cerebral artery (MCA) on this side. At this time, the LDF showed that the MCA-dominated cortical regional blood flow decreased to less than 25% of the blood flow base value, which was considered as successful modeling. After 2 h of obstruction, the suture was slowly pulled out, the transected end of the ECA was ligatured, the CCA artery clamp was released, and the skin was sutured. A small animal heating pad was used to maintain the anal temperature of the rats at 37°C during the opera- tion and 30°C after the operation, and the rats were intraperitone- ally injected with 5 mL of normal saline to prevent dehydration.

After awakening, the rats were put back into the cage, and allowed to take food and water freely. 2.2 Experimental grouping and administration

The experimental animals were randomly divided into 3 groups, with 10 rats in each group. The specific groups were as follows: (1) Model group: After 10 min of cerebral ischemia, the rats were intranasally administered with 150 pL/kg normal saline on both sides. (2) Intravenous baicalin administration group: After 10 min of cerebral ischemia, the tail vein of the rats was injected with a baicalin solution (15 mg/kg). (3) Baicalin-calcium alginate thermosensitive nanogel group:

After 10 min of cerebral ischemia, the rats were intranasally ad-

ministered with the baicalin-calcium alginate thermosensitive nanogel (0.4 mg/kg, 167.7 uL/kg according to the baicalin) on both sides each time. In order to avoid asphyxia caused by simultaneous administration on both sides, the drug was administered to the in- farcted side nasal cavity first and then to the normal side nasal cavity 10 min later. After administration of the drug to both nos- trils, anesthesia was continued for 10 min to allow the gel to so- lidify in the nasal cavity.

A total of 3 doses were given, respectively after 10 min, 24 h and 48 h of cerebral ischemia. After 72 h of cerebral ischemia, the rats were sacrificed and the brains were harvested. 2.3 Evaluation on cerebral infarction volume

After 72 h of cerebral ischemia, the rats were anesthetized with isoflurane, and the brains were harvested after perfusion with cardiac saline solution. The brains were placed in a brain slice mold (the olfactory bulb and cerebellum were removed), and 2 mm thick coronal slices were successively made from front to back, and stained in a 2% TTC solution for 15 min (TTC is a fat-soluble light-sensitive complex and a proton receptor of mitochondrial respiratory chain. The dehydrogenase in normal brain tissue reacts with TTC and turns red. However, the ischemic brain tissue cannot react with TTC due to the decreased activity of mitochondrial de- hydrogenase in cell membrane, showing a white anti-staining area).

The Image J analysis software was used for analysis. The specific formula was as follows:

Cerebral infarction volume (%) = (normal side hemispheric volume - normal volume of the ischemic side hemisphere) + normal side hemispheric volume x 100%. 3. Experimental results 3.1 Effects of the baicalin-calcium alginate thermosensitive nanogel on acute cerebral ischemic infarction volume of rats

After TTC staining, the normal brain tissue of the rats was uniformly red, and the striatum, hippocampus, cortex and other ar- eas of the ischemic hemisphere brain tissue in the model group showed a white anti-staining area (infarct), indicating that the model was successfully made. Compared with the model group, both the nasal administration (0.4 mg/kg) of the baicalin-calcium algi-

nate thermosensitive nanogel and the intravenous administration (15 mg/kg) of the baicalin could reduce the cerebral infarction volume after stroke, and the dosage (0.4 mg/kg) of the baicalin- calcium alginate thermosensitive nanogel administered intranasally was only 1/37.5 of that (15 mg/kg) of the baicalin administered intravenously (FIG. 1 and 2).

The results indicated that the baicalin-calcium alginate thermosensitive nanogel had an obvious protective effect on acute stroke injury by nasal administration. Moreover, the dosage (0.4 mg/kg) of the baicalin-calcium alginate thermosensitive nanogel administered intranasally was only 1/37.5 of that (15 mg/kg) of the baicalin administered intravencusly, indicating that the bai- calin-calcium alginate thermosensitive nanogel had a better effi- cacy with small dosage.

The baicalin-calcium alginate thermosensitive nanogel pre- pared by the present invention is liquid at room temperature and administered through nasal cavity, wherein the temperature of na- sal cavity makes the liquid at room temperature become gelatinous and adhere to the surface of nasal cavity, so that the baicalin is absorbed by the nasal mucosa and quickly reaches the brain by by- passing the blood-brain barrier, so as to play a role of treating acute stroke quickly. The corresponding side effects are reduced while the dosage is decreased. Compared with traditional oral and intravenous preparations for treating stroke, the present inven- tion has the advantages of rapid onset, small dosage and conven- ient use.

The above-mentioned embodiments are used for explaining, ra- ther than limiting, the present invention. Any modifications and changes made to the present invention within the protection scope of the spirit and claims of the present invention should fall into the protection scope of the present invention.

Claims

CONCLUSIONS

A method for preparing baicalin-calcium alginate thermosensitive nanogel, wherein the baicalin-calcium alginate thermosensitive nanogel is prepared from an active pharmaceutical ingredient baicalin and pharmaceutically effective excipients, the method specifically comprising the following steps: {1} preparation of nanogel of baicalin calcium alginate adding liquid paraffin, Span 80 and Tween 80 respectively in a weight ratio of (500 to 200):{5 to 1.5):1, and completely dispersing the mixture in a water bath at 40 up to 60°C and 500 rpm as an oil phase; setting a stirring speed at 1000 rpm and dripping 60 to 300 parts of sodium alginate solution containing baicalin into the oil phase at a flow rate of 0.75 ml/min; upon completion of the dripping, stirring the mixed solution continuously until the water phase is thoroughly mixed with the oil phase; adding 20 to 100 parts of hardener at a flow rate of 0.25 ml/min while stirring at a speed of 1000 rpm; after completion of dripping, continuously stirring the mixture for 1 hour, centrifuging the resulting nanogel at 3,000 rpm for 15 minutes, separating the aqueous phase to obtain a suspension of baicalin-calcium alginate nanogel, and flash-freezing with liquid nitrogen and freeze-drying the baicalin-calcium alginate nanogel suspension; wherein in the sodium alginate solution containing baicalin, a sodium alginate concentration is 1 to 10 mg/ml, a baicalin concentration is 0.1 to 20 mg/ml and the curing agent is a 70% ethanol solution of 0.5 to 5 mg/ml ml is calcium chloride; (2) preparation of baicalin-calcium alginate thermosensitive nancgel adding normal saline to 6 parts by weight of baicalin-calcium alginate nanogel prepared in S1, dispersing the nanogel evenly, adding 1 part by weight of poloxamer 188 (P 188) and 7 parts by weight of poloxamer 407 (P 407) under ice bath condition, and mixing the mixture until all materials are evenly dissolved to obtain baicalin-calcium alginate thermosensitive nanogel.

The method for preparing baicalin-calcium alginate thermosensitive nanogel according to claim 1, wherein the baicalin-calcium alginate thermosensitive nanogel is gelatinous at 30 + 2°C.

The process for preparing baicalin-calcium alginate thermosensitive nanogel according to claim 1, wherein a weight ratio of the liquid paraffin to the Span 80 to the Tween 80 is 333.33:2.33:1; the amount of the sodium alginate solution containing baicalin is 100 parts by weight and the amount of the hardener is 33.33 parts by weight.

A thermosensitive baicalin-calcium alginate nanogel prepared by the preparation method according to any one of claims 1 to 3.

Use of the baicalin-calcium alginate heat-sensitive nanogel according to claim 4 in the preparation of a medicament for the treatment of stroke.