CN108371708A - A kind of oral insulin nanoparticle formulations and preparation method thereof - Google Patents
A kind of oral insulin nanoparticle formulations and preparation method thereof Download PDFInfo
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- CN108371708A CN108371708A CN201810107604.4A CN201810107604A CN108371708A CN 108371708 A CN108371708 A CN 108371708A CN 201810107604 A CN201810107604 A CN 201810107604A CN 108371708 A CN108371708 A CN 108371708A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000000203 mixture Substances 0.000 title claims abstract description 13
- 229940125395 oral insulin Drugs 0.000 title claims abstract description 13
- 238000009472 formulation Methods 0.000 title abstract description 6
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Physics & Mathematics (AREA)
- Endocrinology (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Emergency Medicine (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Inorganic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Hematology (AREA)
Abstract
The invention belongs to pharmaceutical technology field, a kind of oral insulin nanoparticle formulations and preparation method thereof are specifically disclosed.It is the nano combined core that chitosan quaternary ammonium salt and insulin form that the nano particle, which has nucleocapsid, core, and shell is hyaluronic acid or thiolated hyaluronic acid coated in core surfaces.The nano particle of the present invention has and compared with high encapsulation rate and/or drugloading rate and has the function of sticking and penetrating small intestine mucus, improves absorption of the insulin in small intestine site, with high oral administration biaavailability；The insulin nano granular preparation can effectively control blood glucose after oral, and compared to hypodermic injection, change of blood sugar is more steady, has good blood sugar decreasing effect and high bioavilability, can effectively control blood glucose, greatly mitigate the unnecessary pain of diabetic, safely and conveniently.
Description
Technical field
The invention belongs to biomedicine technical field, more particularly, to a kind of oral insulin nanoparticle formulations and
Preparation method and application.
Background technology
Insulin since the advent of the world is always the choice drug of insulin-dependent diabetics.Insulin mainly passes through
Subdermal routes of administration, and prolonged and repeated administration is needed, this causes patient greatly pain and low compliance, while can also make
At a series of safety issue, such as allergic reaction, lipoatrophy or hyperplasia, tolerance.Compared to hypodermic injection
The a series of problems brought, oral medication are considered as the administration route being more suitable for.Oral medication can effectively reduce high pancreas
Island element mass formed by blood stasis is taken precautions against and is increased with the treatment-related weight of whole body insulin, reduces the risk of hypoglycemia, while oral insulin
It is absorbed into portal vein by gastrointestinal tract and directly participates in metabolism of the liver to glucose, can accurately simulate human normal physiology
Approach.However due to insulin degradable denaturation under gastrointestinal physiology environment, and absorption efficiency is very low at small intestine, causes mouth
The bioavilability for taking insulin is extremely low.The nanoparticle system of loading albumen, such as liposome, micella, polymer/albumen are compound
Particle, inorganic particulate etc. can effectively improve the oral availability of insulin, be widely used in oral delivery insulin.
Small intestine epithelium surface covers one layer of slime layer, can stop and quickly remove external particle and harmful substance entrance
Small intestine.After oral load medicine particle reaches small intestine, first having to could be by intestinal absorption across mucous layer barrier.Overcome this barrier master
It to be penetrated by mucus and mucus adsorbs two kinds of strategies to realize.The study found that surface hydrophilic and aobvious electroneutral or electronegativity
Particle and slime layer active force it is small, can effectively pass through slime layer arrive at intestinal epithelial cell.However due to its 'inertia'
Surface nature, particle is difficult to be absorbed by intestinal epithelial cell；And due to the interaction force of its shortage and small intestine mucus, cause
Its small enteral residence time declines；It is relatively low that a series of this reason causes mucus to penetrate system bioavilability.Mucus adsorptivity material
Material can increase particle and enter mucus by electrostatic force, hydrogen bond, hydrophobic force, the modes such as covalent bonding and slime layer interaction
Residence time of the efficiency and particle of layer at small intestine promotes absorption of the insulin at small intestine.However, since mucus adsorbs
Property particle and mucus there is stronger active force, particle is easy to be trapped in slime layer, and is quickly removed by slime layer, can not reach
At small intestine epithelium, cause the bioavilability of insulin not high.Still lack one kind now and effectively overcomes small intestine mucous layer barrier
Insulin delivery system.
Invention content
The technical problem to be solved by the present invention is to overcome defect and deficiency existing for existing insulin delivery system, pass through
A kind of loaded with nucleocapsid, which has been prepared, using modified chitosan quaternary ammonium salt and thiolated hyaluronic acid pancreas islet
The nano particle of element, the nano particle has the function of sticking and penetrating small intestine mucus, improves insulin in small intestine site
Absorption, there is high oral administration biaavailability, type-1 diabetes mellitus patient can be given to provide a kind of convenience, no pain is safe and efficient
Administering mode.
The first purpose of the invention is to provide a kind of nano particles of load insulin.
Second object of the present invention is to provide the preparation method of the load insulin nano particle.
Third object of the present invention is to provide the applications of the load insulin nano particle.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
A kind of nano particle of load insulin, it is chitosan quaternary ammonium salt and pancreas islet that the nano particle, which has nucleocapsid, core,
The nano combined core of element composition, shell are hyaluronic acid or thiolated hyaluronic acid coated in core surfaces.
Chitosan quaternary ammonium salt in nano particle of the present invention after quaternised modified has good water in physiological conditions
Dissolubility, while having and preferably opening close-connected ability；And hyaluronic acid good biocompatibility, the mercapto after sulfhydryl modified
Base hyaluronic acid can form disulfide bond with mucoprotein in small intestine mucus, enhance mucus adsorptivity.
Preferably, the molecular weight of the chitosan quaternary ammonium salt is 50kDa~200kDa (preferably 50kDa), quaternized journey
Degree is 10%~70%（Preferably 43%）；The molecular weight of thiolated hyaluronic acid is 3.4kDa~200kDa (preferably 35kDa),
Sulfhydrylation degree is 5%~30%（Preferably 12.3%）.
Preferably, the grain size of the nano particle is 50~200nm（Preferably 100nm）.
Preferably, the PDI of the nano particle is 0.1~0.2.
Preferably, the current potential of the nano particle is -5 mV of mV~-30.
Preferably, the drugloading rate of the nano particle is 25%~60%.
The nano particle of load insulin of the present invention has the function of sticking and penetrating small intestine mucus, improves insulin and exist
The absorption of small intestine site has high oral administration biaavailability；Therefore the present invention is also claimed above-mentioned nano particle and is preparing
Application in Macrulin.
A kind of oral insulin medicament preparation, includes the nano particle of above-mentioned load insulin.
Preferably, the pharmaceutical preparation also includes pharmaceutically acceptable excipient.
Preferably, the pharmaceutical preparation is lyophilized preparation.
Preferably, the pharmaceutical preparation is capsule.
A method of load insulin nano particle is prepared, is included the following steps：
S1. by chitosan quaternary ammonium salting liquid by channel 1 and 2, insulin solutions are reached in vortex mixing region and are mixed by 3 and 4
It closes, obtains the nano combined core of insulin and chitosan quaternary ammonium salt composition；Four channel liquids at the uniform velocity flow, flow velocity 2mL/
Min~50mL/min（Preferably 5mL/min~50mL/min, more preferably 40mL/min）；
S2. by mixed liquor obtained by S1, by channel 1 and 2, hyaluronic acid or thiolated hyaluronic acid solution are arrived by channel 3 and 4
Up to being mixed in vortex mixing region, surface coated with hyaluronic acid or thiolated hyaluronic acid nano particle are obtained；Four channel liquid
Body at the uniform velocity flows, and flow velocity is 2mL/min~50mL/min（Preferably 5mL/min~50mL/min, more preferably 40mL/min）；
Preferably, the present invention prepares load insulin nano particle using FNC technologies in multiple entry vortex mixer, described
Multiple entry vortex mixer（Such as Figure 1A, shown in 1B）Including the superposed first component, the second component positioned at middle part and position
Third member in lower part, the first component, second component and third member are the cylinder with same diameter；First
Part is provided with multiple channels（Preferably 4, respectively channel 1, channel 2, channel 3, channel 4）, second component setting is vortexed mixed
Region and multiple water conservancy diversion regions are closed, channel is arranged in third member；The channel of the first component and the water conservancy diversion regional fluid of second component
Connection；The water conservancy diversion region of second component is connected to vortex mixing regional fluid；The vortex mixing region of second component and third
The passage of component；With high throughput, the strong feature of controllability, nano particle obtained be evenly distributed and grain size compared with
Small, difference is small between batch；Above-mentioned technology and device are documented in the present inventor, and application No. is the special of PCT/US2017/014080 early period
In profit.
Preferably, the pH of the insulin solutions is 7~9（It is preferred that 8.0）；Insulin is specially dissolved in pH=2
In HCl solution, after with NaOH solution pH value is adjusted to 7~9（It is preferred that 8.0）.
Preferably, the quaternization degree of the chitosan quaternary ammonium salt is 10%~70%（Preferably 43%）；The sulfhydrylation is saturating
The sulfhydrylation degree of bright matter acid is 5%~30%（Preferably 12.3%）.
Preferably, a concentration of 0.2~3mg/mL of the chitosan quaternary ammonium salt（Preferably 1mg/mL）, insulin solutions are dense
Degree is 0.5~3mg/mL（Preferably 2mg/mL）.
Preferably, a concentration of 0.25~1mg/mL of the hyaluronic acid or thiolated hyaluronic acid（Preferably 1mg/
mL）.
Preferably, the mass ratio of the insulin and chitosan quaternary ammonium salt is 0.5~2.
Preferably, the preparation method of the chitosan quaternary ammonium salt is：It dissolves the chitosan in the acetum of 2wt%, adds
For heat to 80 DEG C, backward solution is interior to be added dropwise chlorination glycidyltrimetiiylammonium ammonium（GTMAC）Aqueous solution is reacted for 24 hours at 80 DEG C, is obtained
Final product chitosan quaternary ammonium salt（HTCC）.
Preferably, the preparation method of the thiolated hyaluronic acid is：By hyaluronic acid（HA）It is dissolved in distilled water, adds
Enter ion exchange resin stirring 0.5h, filter off resin, solution ph is adjusted to 7.04 using tetrabutylammonium hydroxide, freeze-drying obtains
To product HA-TBA.First step products therefrom HA-TBA is dissolved in DMSO, and 4-dimethylaminopyridine is added, two thio two
Propionic acid, di-tert-butyl dicarbonate react 20h at 45 DEG C.To water dialyse, remove DMSO, after precipitate in acetone twice, be dissolved in
In distilled water, freeze-drying.Lyophilized products are dissolved in distilled water, pH to 8.5 is adjusted, dithiothreitol (DTT) is added, 3h are reacted, by pH
Value is adjusted to 3.5, precipitates once, is redissolved in distilled water in ice ethyl alcohol, freeze-drying obtains thiolated hyaluronic acid（HA-
SH）.
The present invention is thiolated modified by being carried out to mucus penetrability material-hyaluronic acid, obtains thiolated hyaluronic acid,
And thiolated hyaluronic acid is coated on using FNC technologies and loads insulin nano particle surface, it can be with small intestine mucus
Layer has compared with strong interaction, compared to merely using hyaluronic acid as coat, increase particle enter small intestine slime layer with
And the residence time at small intestine；Meanwhile into after small intestine slime layer, thiolated hyaluronic acid coating can gradually drop at any time
Solution falls off, and has better mucus penetrability compared to the nano combined core of simple insulin and chitosan quaternary ammonium salt, subtracts
It is trapped in the nano particle of slime layer less, increases the particle reached at small intestine epithelium；Particle reaches small intestine epithelium across slime layer
During this, outer layer thiolated hyaluronic acid coat falls off, and has the chitosan for opening the close linkage function of small intestine epithelium
Quaternary ammonium salt is exposed, and plays a role, and promotes insulin by cell bypass by intestinal absorption.Insulin nano system in the present invention
Agent can effectively control blood glucose after oral, and compared to hypodermic injection, change of blood sugar is more steady, have good hypoglycemic
Effect and high bioavilability.The Macrulin of the present invention can effectively control blood glucose, greatly mitigate glycosuria
The unnecessary pain of patient, safely and conveniently.
Compared with prior art, the invention has the advantages that：
Nano particle of the present invention is with compared with high encapsulation rate and/or drugloading rate and with the work(for sticking and penetrating small intestine mucus
Can, absorption of the insulin in small intestine site is improved, there is high oral administration biaavailability；The insulin nano preparation, mouth
Blood glucose can be effectively controlled after clothes, and compared to hypodermic injection, change of blood sugar is more steady, have good blood sugar decreasing effect with
And high bioavilability, blood glucose can be effectively controlled, the unnecessary pain of diabetic, safety and side are greatly mitigated
Just.
Description of the drawings
Fig. 1 is to be illustratively described the multiple entry vortex mixer for being used to prepare the nanoparticle of the present invention；Figure 1A is
After one component, second component and third member assembling and it is connected to the state of external pipe；Figure 1B -1 is looking up for the first component
Figure；Figure 1B -2 is the vertical view of second component；Figure 1B -3 is the vertical view of third member；Fig. 1 C show in embodiment 1 and are used for
The device of nanoparticle is prepared, Fig. 1 C-1 show that syringe, high-pressure pump, plastic tube and multiple entry vortex mixer, Fig. 1 C-2 are
It is connected to the enlarged drawing of the multiple entry vortex mixer of plastic tube.
Fig. 2 show the schematic diagram of multiple entry vortex mixer and what the present invention prepared nano particle prepare schematic diagram.
Fig. 3 show obtained nano combined core different in flow rate in embodiment 3（NC）.A concentration of 1mg/mL of HTCC, pancreas
Island a concentration of 2mg/mL of element.
Fig. 4 show the nanometer core that different insulin and HTCC ratios obtain in embodiment 3（NC）, flow velocity 40mL/
A concentration of 1mg/mL of min, HTCC.
Fig. 5 show the encapsulation rate and drugloading rate of corresponding NC under different insulin/HTCC ratios in embodiment 3.
Fig. 6 show NP obtained in embodiment 3HAAnd NPHA-SH, flow velocity 40mL/min.
Fig. 7 show NC obtained, NP under optimum conditionHAAnd NPHA-SHTransmission electron microscope picture.
Fig. 8 show the insulin releasing curve of Imitative gastroenteric environments in embodiment 5.
Fig. 9 show in embodiment 5 insulin releasing curve under physiological condition.
Figure 10 show mucoprotein in embodiment 6, and HA-SH/ mucoproteins blend and HA-SH/ mucoproteins are lyophilized after being incubated
The DSC curve of powder.
Figure 11 show NC, NP in embodiment 7HAAnd NPHA-SHFluorescent image in photobleaching process.
Figure 12 show photobleaching region average fluorescent strength in embodiment 7 and changes over time relationship.
Figure 13 show NP in embodiment 8HAAnd NPHA-SHDistribution in E12 monolayers, wherein HTCC RITC
Label, insulin are marked with Cy5.
Figure 14 show NP in embodiment 8HAAnd NPHA-SHDistribution in E12 monolayers, wherein HA and HA-SH are used
Rhodamine 123 marks, and insulin is marked with Cy5.
Figure 15 is shown in embodiment 9 after three kinds of particle disposals, removes the fluorescent image of small intestine before and after mucus.
Figure 16 is shown in embodiment 9 after three kinds of particle disposals, and the mean fluorecence on small intestine surface is strong before and after removing mucus
Degree.
Figure 17, which is shown in rear embodiment 10, is added the change of insulin solutions or nano particle cell transmembrane resistance at any time
Change.
Figure 18 show the apparent infiltration system that variable grain transport insulin in embodiment 10 passes through caco2 monolayers
Number.
Figure 19 show in embodiment 10 and NP is addedHA-SHThe close connection of caco2 cells before and after particle.
After Figure 20 show oral insulin solution in embodiment 11 or carries insulin granule, insulin is in small intestine site
Absorbing state.
Figure 21 show the hypoglycemic song taken orally in embodiment 12 after carrying insulin granule or subcutaneous insulin injections solution
Line
Figure 22, which is shown in embodiment 13, takes orally NPHA, NPHA-SH, the blood after insulin solutions and subcutaneous insulin injections solution
Concentration changes over time.
Figure 23 show the NC nano particles obtained using the HTCC of different quaternization degrees in embodiment 14.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
1 chitosan quaternary ammonium salt of embodiment（HTCC）Synthesis
2g chitosans are dissolved in the acetum of the 2wt% of 100mL, are heated to 80o5mL chlorine is slowly added dropwise after C into solution
Change glycidyltrimetiiylammonium ammonium（GTMAC）Aqueous solution, 80oIt is reacted under C for 24 hours, the third of 10 times of volumes after acquired solution cooling
It is precipitated 2 times in ketone, sediment redissolves in distilled water, dialyses 3 days to water, and freeze-drying obtains final product HTCC.Wherein GTMAC
Aqueous solution mass concentration is that 20%, HTCC quaternization degrees are 43%.In addition, being that 2mL or 4mL can by adjusting the volume of GTMAC
It is respectively the HTCC for 12% and 23% to obtain quaternization degree.
2 thiolated hyaluronic acid of embodiment（HA-SH）Synthesis
1, by hyaluronic acid（HA）It is dissolved in distilled water, a concentration of 5mg/mL, ion exchange resin is added（Resin and hyalomitome
Sour mass ratio is 3：1）, 0.5h is stirred, resin is filtered off, solution ph is adjusted to 7.04 using tetrabutylammonium hydroxide, freeze-drying obtains
To product HA-TBA.
2, by HA-TBA（1.5g）It is dissolved in DMSO, a concentration of 0.5mg/mL, and 4-dimethylaminopyridine is added
（0.35g）, dithiodipropionic acid（2.4g）, di-tert-butyl dicarbonate（0.2mL）, 20h is reacted at 45 DEG C.After being cooled to room temperature,
To distilled water dialyse, remove DMSO, after precipitated in the acetone of 10 times of volumes twice, be dissolved in distilled water, to water dialysis 3
It, freeze-drying.By lyophilized products（0.5g）It is dissolved in distilled water, adjusts pH to 8.5, dithiothreitol (DTT) is added（0.5g）, reaction
PH value is adjusted to 3.5 by 3h, is precipitated once, is redissolved in distilled water in ice ethyl alcohol, freeze-drying obtains thiolated hyaluronic acid
（HA-SH）.The sulfhydrylation degree of HA-SH is 12.3%.Wherein, the quality for adjusting dithiodipropionic acid is 1.5g and 5.0g, can be with
Obtain the HA-SH that sulfhydrylation degree is respectively 8.4% and 32.3%.
Embodiment 3 prepares nano particle using FNC technologies
FNC technologies are to prepare the technology of drug-loading nanoparticles in a kind of water phase by electrostatic interaction（It is documented in the present inventor
Early period, application No. is in the patent of PCT/US2017/014080）, preparation process mainly carries out in multiple entry vortex mixer,
With high throughput, the strong feature of controllability, nano particle obtained is evenly distributed and grain size is smaller, and difference is small between batch.Due to
The participation of organic solvent-free in whole process, particularly suitable for the formulation of the large biological molecules such as albumen, nucleic acid.The multiple entry
Vortex mixer（Figure 1A, Figure 1B）Second component including the superposed first component, positioned at middle part and positioned at lower part
Three components, the first component, second component and third member are the cylinder with same diameter；The first component is provided with more
A channel（Preferably 4, respectively channel 1, channel 2, channel 3, channel 4）, vortex mixing region and more is arranged in second component
Channel is arranged in a water conservancy diversion region, third member；The channel of the first component and the water conservancy diversion region of second component are in fluid communication；Second
The water conservancy diversion region of part is connected to vortex mixing regional fluid；The vortex mixing region of second component and the channel of third member are flowed
Body is connected to.
1, the preparation of insulin and the nano combined cores of HTCC
HTCC is dissolved in the water, a concentration of 1mg/mL.Insulin is dissolved in the HCl solution of pH=2, after with NaOH solution by pH
Value is adjusted to 8.0, a concentration of 0.5~3mg/mL of insulin solutions.HTCC solution and insulin solutions are respectively charged into four injections
In device, four syringes are respectively placed on high-pressure pump, the injection orifice of each syringe respectively with 1~4 respective one end of plastic tube
It is tightly connected, the other end of plastic tube is close by connecting component and four channels of the multiple entry vortex mixer first component respectively
Envelope connection.The first component, the second component of multiple entry vortex mixer are connected with third member by bolt seal, and third
The channel of component is tightly connected by one end of connecting component and plastic tube 5, and the other end of plastic tube 5 connects collection vessel；Figure
1C shows that the device for preparing nanoparticle of the present embodiment, Fig. 1 C-1 show syringe, high-pressure pump, plastic tube and multiple entry whirlpool
Mixer is flowed, Fig. 1 C-2 are the enlarged drawing for the multiple entry vortex mixer for being connected to plastic tube.
High-pressure pump is opened, makes HTCC solution and insulin solutions and insulin solutions simultaneously with the flow velocity of 25mL/min through modeling
Expects pipe 1-4 enters multiple entry vortex mixer, and is mixed in the vortex mixing region of second component；The HTCC solution is distributed in
First and second channel, insulin solutions be distributed in third and fourth lane, as shown in Fig. 2, four channel velocities are consistent, adjust
Section channel velocity, 2mL/min, 5 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min, 50mL/min,
Make two kinds of solution pass through four channels arrival vortex mixing area to be mixed, obtains insulin and the nano combined core of HTCC
（NC）.
Fig. 3 show obtained NC different in flow rate.HTCC a concentration of 1mg/mL, insulin concentration 2mg/mL.Work as flow velocity
When less than 20mL/min, grain diameter reduces with flow velocity and is increased, when flow velocity is between 20~50mL/min, grain diameter
It is constant.When flow velocity is 40mL/min, obtained particle is the most uniform.Select this optimum condition of flow velocity 40mL/min.
A concentration of 1mg/ of the NC that Fig. 4 show different insulin and HTCC ratios obtain, flow velocity 40mL/min, HTCC
mL.As insulin/HTCC（w/w）When more than 2, particle current potential drastically declines, and grain size increased dramatically；As insulin/HTCC（w/w）
When between 0.5~2, obtained particle current potential and grain size is essentially identical.
Fig. 5 show the encapsulation rate and drugloading rate of corresponding NC under different insulin/HTCC ratios.It is higher in order to obtain
Drugloading rate, final choice insulin concentration be 2mg/mL, this optimum condition of a concentration of 1mg/mL of HTCC.
2, surface applies HA（NPHA）Or HA-SH（NPHA-SH）The preparation of nano particle
Hyaluronic acid or thiolated hyaluronic acid are dissolved in the water, a concentration of 0.25mg/mL, 0.5mg/mL, 0.75mg/mL,
1mg/mL, NC suspensions made from the first step are distributed in first and second channel, HA HA-SH aqueous solutions be distributed in third and
Fourth lane, as shown in Figure 2 B, four channel velocities are consistent, adjust channel velocity 5 mL/min, 10 mL/min, 20 mL/
Min, 30 mL/min, 40 mL/min, 50mL/min make two kinds of liquid pass through four channels arrival vortex mixing region and are mixed
It closes, obtains surface coating HA（NPHA）Or HA-SH（NPHA-SH）Nano particle.
Table 1 is the NP that is obtained under optimum condition after screeningHAAnd NPHA-SH。
Fig. 6 show NP obtainedHAAnd NPHA-SH, flow velocity 40mL/min.NC through the surfaces HA or HA-SH apply after,
Grain size increases, and charge inverts.For two different coatings, the two grain size and current potential are essentially identical.When HA or HA-SH are dense
When degree gradually rises, grain size is gradually reduced, while current potential gradually increases.A concentration of 1mg/mL of final choice HA and HA-SH, at this time
There are minimum grain size and polydispersity index.Table 1 show NP obtained under optimum conditionHAAnd NPHA-SH。
NP obtained under 1 optimum condition of tableHAAnd NPHA-SH
| Nano particle | Grain size（nm） | PDI | Zeta-potential（mV） | Encapsulation rate（%） | Drugloading rate（%） |
| NPHA | 101±3 | 0.11±0.03 | -25.3±2.0 | 90.5±0.6 | 37.6 ± 0.2 |
| NPHA-SH | 102±3 | 0.11±0.02 | -26.2±1.0 | 91.1±0.4 | 38.0 ± 0.1 |
4 grain size of embodiment, current potential and Morphological Characterization
Grain size, polydispersity index and the surface potential of particle are measured using Malvern particle instrument.It is characterized using transmission electron microscope
The form of particle.
Fig. 7 show NC obtained, NP under optimum conditionHAAnd NPHA-SHTransmission electron microscope picture.The grain size of three kinds of particles with
The grain size that Malvern particle instrument obtains is consistent.NP is clear that from figureHAAnd NPHA-SHThe nucleocapsid of particle.
5 vitro drug release of embodiment is tested
NC suspension is diluted 1 times for use.Take the NC particle suspension liquids of one times of the dilution of 1mL, the NP of 1mLHASuspension and
The NP of 1mLHA-SHParticle suspension liquid is respectively placed in the dialysis tubing of 1mL, sealing.The PBS that dialysis tubing is placed in 20mL 10mM delays
It in fliud flushing, is incubated in 37 DEG C, the shaking table of 150rpm, 1mL buffer solutions is taken out every specific time（It is then slow with the blank of 1mL
Fliud flushing is supplemented, and system constancy of volume is kept）, insulin concentration is measured using BCA detection methods.
（1）The variation of pH value when particle passes through gastrointestinal system is simulated, stomach pH value is 2.5, and the gastric emptying time is 2~3h,
Small intestines pH value is 6.8~7.4, and in order to simulate the pH environment of gastrointestinal system, dialysis tubing is first placed in the HCL solution of pH=2.5
In, it is primary every 1h samplings.It is primary every 2h samplings by the PBS buffer solution for the 10mM that buffer exchange is pH=6.8 after 2h.
Fig. 8 show the release profiles of insulin.In stomach pH environment, after 2 hours, NC particles have 50% insulin releasing
Out, however for HA and HA-SH the particle NP coatedHAAnd NPHA-SHOnly 33% insulin releasing comes out, and thus illustrates
The particle of HA or HA-SH claddings has better protecting effect in stomach to insulin.
（2）Dialysis tubing is placed in the PBS buffer solution of the 10mM of pH=7.4, it is primary every 2h samplings.Fig. 9 show physiology
Under the conditions of insulin releasing curve.In physiological conditions, in 24 hours, the insulin higher than 70% can be released.
Embodiment 6 utilizes differential scanning calorimetry（DSC）Measure HA-SH and mucoprotein（mucin）Interaction
HA-SH and pig mucoprotein are dissolved in the PBS solution of 10mM altogether, 37oIt is incubated 8 hours under C, it is rear to be lyophilized.It is surveyed using DSC
Mucoprotein is measured, the endothermic curve of freeze-dried powder after HA-SH/ mucoproteins blend and HA-SH/ mucoproteins are incubated.Wherein HA-SH with it is viscous
The weight ratio of albumen is 1:1, heating rate 20oC/min。
As shown in Figure 10, mucoprotein is 230oIt is melted when C, there is apparent endothermic peak；HA-SH/ mucoprotein blends exist
230oC is still it is observed that the melting peak of apparent mucoprotein；However after the incubation of HA-SH/ mucoproteins in the DSC curve of freeze-dried powder,
This endothermic peak disappears, it was demonstrated that mucoprotein is combined with HA-SH completely.
Embodiment 7 measures the movement for carrying insulin nano particle in mucus using photobleaching
By 20 μ L nano granule suspensions（Insulin（INS）A concentration of 0.5mg/mL, insulin are marked with RITC）It is small with 60 μ L
Intestines mucus is uniformly mixed, 37oIt is incubated 0.5h under C, is placed on fluorescence microscope coverslip, utilizes laser confocal microscope
A certain specific region is exposed to 100% fluorescence intensity（Excitation wavelength：552nm）Lower 60s, and acquire primary figure every 10s
Picture, and count the average fluorescent strength of exposure area.
NC shown in Figure 11, NPHAAnd NPHA-SHFluorescent image in photobleaching process.Figure 12 show photobleaching region
Average fluorescent strength changes over time relationship.The fluorescence intensity of photobleaching region depend on photobleaching process fluorescence losses and
The fluorescence that nano particle free movement generates restores.After photobleaching, fluorescence intensity loss is smaller, and particle locomitivity is stronger.60s
Afterwards, NPHAFluorescence losses only~17% have strongest locomitivity.NPHA-SHFluorescence losses and NPHAIt is close, have similar
Locomitivity, NC fluorescence losses are maximum ,~50%, locomitivity is worst in mucus.
Distribution of 8 drug-loading nanoparticles of embodiment in E12 monolayers
E12 monolayers are used for simulating the small intestine epithelium system with slime layer.E12 cell inoculations are incubated in cavate coverslip
It educates 3 days, E12 cells form cell monolayer layer.By 100 μ LNPHAAnd NPHA-SHIt is co-cultured in E12 cells, it, will after being incubated 2 hours
Particle takes out, and is cleaned 3 times with PBS, fixes cell using 4% paraformaldehyde solution, DAPI is used in combination to dye nucleus,
It is placed on the fluorescent image that different depth is observed and acquired under laser confocal microscope.Wherein insulin is marked with Cy5, HTCC
The distribution of the nano combined cores of HTCC/INS is measured with RITC labels；Insulin is marked with Cy5, HA and HA-SH Luo Dan
Bright 123 mark to measure insulin and HA（HA-SH）Distribution.
Figure 13 show insulin solutions, NC, NPHAAnd NPHA-SHDistribution in E12 monolayers, wherein HTCC
It is marked with RITC, insulin is marked with Cy5.NC particles are in 30 μm and 15 μm of depth（Slime layer）It observes stronger
The signal of HTCC and insulin, and the two common location.NPHAAnd NPHA-SH30 μm of depth do not observe substantially HTCC and
The signal of INS, when depth is 15 μm, it is observed that the signal of faint HTCC and insulin, and the two common location.
Depth is 0 μm（Cellular layer）Three kinds of particles are it is observed that the signal of strong HTCC and INS, and the two common location.Thus it proves
The positive electricity core of HTCC and INS keeps combining during across slime layer, does not dissociate, and NPHAAnd NPHA-SHCompare NC
With stronger mucus penetration capacity.It is NP at 15 μm in depthHA-SHCompared to NPHAWith stronger signal, illustrate NPHA-SH
There is stronger interaction with slime layer.
Figure 14 show NP in embodiment 8HAAnd NPHA-SHDistribution in E12 monolayers, wherein HA and HA-SH are used
Rhodamine 123 marks, and insulin is marked with Cy5.It is not observe the signal of INS, HA and HA-SH at 30 μm in depth.It is deep
When degree is 15 μm, the two observes faint insulin signaling and the Shell Materials for being uniformly distributed in whole region（HA or
HA-SH）Signal, and not with the signal common location of insulin；When depth is 0 μm, two kinds of particles observe strong insulin
With Shell Materials（HA and HA-SH）Signal, most of HA and HA-SH not with insulin common location, it was demonstrated that NPHAAnd
NPHA-SHParticle effectively can pass through slime layer to reach cell, and during across slime layer, HA and HA-SH shell meetings
It gradually splits away off, after reaching cellular layer, positively charged core can be exposed.
Embodiment 9 carries distribution of the medicine particle at small intestine mucus
SD Rat Fasts 12 hours after anesthesia, by operation, ligature at jejunum in rats by both ends to form closing for one section of 5cm
Region is closed, and injects the load medicine particle of 0.5ml, wherein insulin is marked with Cy5, and after 2 hours, this enclosed region is taken out,
Radially cut off, cleaned 3 times with the PBS of 10mM, after its slime layer is fixed upward, in small animal living body imager observation at
Picture is afterwards siphoned away small intestine surface slime layer using vacuum pump, is again placed in small animal living body imager and is observed imaging, and counts
The average fluorescent strength of intestinal regions before and after removing slime layer.
Figure 15 is shown after three kinds of particle disposals, removes the fluorescent image of small intestine before and after mucus.Figure 16 show statistics
Gained removes the average fluorescent strength on small intestine surface before and after mucus.The small intestine that NC is handled before removing mucus has strongest insulin
Signal, NPHA-SHTake second place, NPHAIt is most weak.After removing slime layer, NPHA-SHThe small intestine of processing has strongest insulin signaling, NPHA
Take second place, NC is worst.Remaining insulin as successfully penetrates the insulin of small intestine slime layer after removing mucus.Prove NPHA-SHTool
There are good mucus adsorption property and mucus to penetrate property.
10 cell penetration test of embodiment
To simulate intestinal epithelial cell layer on caco2 cell monolayers, 1% mucoprotein solution simulates small intestine epithelium slime layer.
In transwell plates, 37 DEG C are cultivated 2~3 weeks Cacao2 cell inoculations, until caoco2 cells form one layer of cell monolayer layer
And cell transmembrane resistance is higher than 700 Ω, and close connection is formed between cell,.The culture medium above and below cell membrane is replaced before experiment
For HBSS.The mucoprotein solution of addition 1% is to simulate small intestine epithelium mucus on cellular layer.
（1）Cellular layer cross-film resistance：By 200 μ L insulin solutions, NC, NPHAAnd NPHA-SHParticle suspension liquid is thin with caco2
Born of the same parents are incubated 2 hours altogether, and particle is taken out, and continue to be incubated 8 hours.Every specific time, cell transmembrane resistance is measured.
After Figure 17 show addition insulin solutions or nano particle, cell transmembrane resistance changes with time.For pancreas
Island element solution, cell transmembrane resistance are basically unchanged；For other particles, after particle is added, cell transmembrane resistance declines；It takes out
After particle, cell transmembrane resistance value is gradually restored, it was demonstrated that the intercellular close link of opening that experiment particle can be reversible.For
Without mucus caco2 cellular layers, NC particles make resistance value decline 50% or so, and there is best close connection to open effect, NPHAOr
NPHA-SHParticle makes resistance value drop to 40% or so.For having mucus caco2 cellular layers, NPHAOr NPHA-SHCan still it make thin
Born of the same parents' resistance declines 37% or so, and NC is only capable of that cell resistance is made to decline 30%.Electropositive NC particles and electronegative mucoprotein phase
Interaction is hindered and is played a role at NC particles arrival cellular layer.HA overlays have elecrtonegativity, prevent NPHAParticle and viscous egg
White interaction, therefore close-connected capacity very little is opened it in the addition of mucoprotein.For NPHA-SHAlthough HA-SH
The sulfydryl of coat can form disulfide bond with mucoprotein, but since HA-SH overlays can be under particle surface be gradually removed
Come so that NPHA-SHParticle can be detached from the obstruction of mucoprotein, and mucoprotein opens close-connected ability also substantially without shadow to particle
It rings.
（2）Apparent permeability coefficients measure：The different particle of insulin solutions and 3 is incubated 4h altogether with caco2 cells respectively,
100 μ L solution are taken out from transwell cavity of resorptions every 1h, and the HBSS for supplementing same volume measures fluorescence intensity, passes through formula
（1）Papp values are calculated.
In formula,PappFor apparent permeability coefficients, dQ/dtFor flow of the particle from transwell epicoele to cavity of resorption of fluorescent marker,C o For the initial fluorescent intensity of epicoele,AFor transewell membrane areas.
Figure 18 show the apparent permeability coefficients that variable grain transport insulin passes through caco2 monolayers.For without viscous
The caco2 cellular layers of albumen, NC have highest apparent permeability coefficients.For there is the caco2 cellular layers of mucus, compared to NC,
NPHAAnd NPHA-SHWith higher apparent permeability coefficients.After mucoprotein is added, NC apparent permeability coefficients drastically decline, and viscous egg
White addition, to NPHAAnd NPHA-SHApparent permeability coefficients almost without influence.Compared with NC, NPHAAnd NPHA-SHWith more preferable
Mucus penetration capacity, can more effectively promote absorption of the small intestine to insulin.
（3）Close connection imaging：Using Occludin antibody as primary antibody, it is small that it is incubated 0.5 altogether with caco2 cells at room temperature
When, so that it is fully combined with close connected Occludin, the rear secondary antibody that AF488 labels are added is incubated 1 hour at room temperature,
Secondary antibody is set fully to be combined with primary antibody.Under fluorescence microscope, NP is added in observationHA-SBefore H particles, with NPHA-SHParticle is incubated two altogether
After hour, and continue the intercellular close connection after being incubated 10 hours after taking out particle.
Figure 19, which is shown, is added NPHA-SHThe close connection of caco2 cells before and after particle.Before particle is added（0h）It can observe
To continuously clearly cyclic structure；NPHA-SH is added, after being incubated 2h altogether, cyclic structure disappears, and illustrates closely to connect and open；It moves
Except particle, continue to be incubated 10h, and be observed that continuous clearly cyclic structure, illustrating closely to connect can reply.Thus it says
It is bright the present invention in particle can reversible opening closely connect, promote insulin by cell bypass by intestinal absorption.
The oral absorption for carrying insulin nano particle in small intestine site of embodiment 11
SD Rat Fast 12h take orally insulin solutions, NC, NP afterwardsHAAnd NPHA-SHParticle, wherein insulin are marked with RITC,
After 2 hours, rat anesthesia is put to death, by operation, takes out the jejunum position of small intestine, is sliced, is fixed with 4% paraformaldehyde small
Intestinal tissue dyes intestinal epithelial cell core and small intestine slime layer using DAPI and AF-645, in laser co-focusing
It is imaged under microscope.
After Figure 20 show oral insulin solution or carries insulin granule, absorbing state of the insulin in small intestine site.
Oral NPHA-SHGroup has strongest absorption of insulin, while still with there is a large amount of insulin in slime layer；Oral NPHAGroup has
Stronger absorption of insulin；Insulin signaling is observed in oral NC slime layers, and only small part insulin enters small intestine
Epithelial cell, oral absorption are poor；Oral insulin solution group does not observe insulin signaling substantially.
12 oral hypoglycaemic effect of embodiment
The SD rats of 200~250g become type-1 diabetes mellitus model mouse by the way that the induction of 80mg/kg streptozotocins is injected intraperitoneally.It will
Model mouse is divided into 6 groups, every group 7.Before experiment, 10~12h of all experimental group fasting organizes 1 gavage deionized water, organizes 2 gavage pancreases
Island element solution（75IU/kg）, organize 3 gavage NC particle suspension liquids（75IU/kg）, organize 4 gavage NPHAParticle suspension liquid（75IU/kg）,
5 gavage NP of groupHA-SHParticle suspension liquid（75IU/kg）, organize 6 subcutaneous insulin injections solution（5IU/kg）.Every 1h, tail point takes
Blood measures blood glucose using blood glucose meter.
Figure 21 show the oral hypoglycemic curve carried after insulin granule or subcutaneous insulin injections.Pancreas islet is subcutaneously injected
Plain experimental mouse blood glucose is dropped rapidly to reduced levels, and oral insulin nanoparticle formulations blood glucose can smoothly decline.
NPHA-SHWith strongest blood sugar decreasing effect, NPHATake second place, NC blood sugar decreasing effects are poor.Oral insulin solution is as negative
Control, on blood glucose substantially without influence.
Embodiment 13NPHAAnd NPHA-SHOral administration biaavailability
The type-1 diabetes mellitus model mouse of 200~250g is divided into 4 groups, 10~12h of all experimental group fasting before testing.Group 1 is subcutaneously noted
Penetrate insulin solutions（5IU/kg）, organize 2 gavage NPHAParticle suspension liquid（75IU/kg）, organize 3 gavage NPHA-SHParticle suspension liquid
（75IU/kg）, organize 4 gavage insulin solutions（75UI/kg）.Every specific time, eye socket takes blood, is tried using insulin ELISA
Agent box measures the concentration of insulin in blood, obtains the curve of insulin blood concentration and time, as shown in figure 22.It is subcutaneously injected
Insulin group can be given birth to as 100% control by comparing oral granule group and subcutaneous insulin injections group area under the curve
Object availability.NPHAAnd NPHA-SHOral administration biaavailability be respectively：5.8% and 11.3%.Oral insulin solution group conduct
Negative control, insulin concentration is close to zero.
Influence of the 14 chitosan quaternary ammonium degree of embodiment to the nano particle of load insulin
Utilize the HTCC of three kinds of different quaternization degrees（12%, 23% and 43%）It is prepared for the NC nanometers of load insulin respectively
Grain, other raw materials and method are same as Example 3.By changing the quaternization degree of chitosan, NC nano particles can be regulated and controled
Size and surface charge.As shown in figure 23, as chitosan quaternary ammonium degree reduces, NC grain diameters increase from 75nm to about
The surface potential of 263nm, particle reduce to about+17mV by+27mV.
The preparation of the nano particle of the sulfhydrylation degree load insulin of 15 hyaluronic acid of embodiment
Using sulfhydrylation degree it is 8.4%, 12.3%, 32.3% thiolated hyaluronic acid prepares the NP of load insulinHA-SH
Nano particle, other raw materials and preparation method are same as Example 3.Since the thiolated modified of HA mainly utilizes in this patent
Esterification occurs for the hydroxyl of HA and the carboxyl of dithiodipropionic acid, and modified HA-SH has electrification similar with HA before modified
Property, thus different sulfhydrylation degree modification hyaluronic acid by charge effect coated in can speculate to obtain after NC nano grain surfaces
The nano particle of grain size, dispersion degree, encapsulation rate and the similar load insulin of drugloading rate.
Claims (10)
1. a kind of nano particle of load insulin, which is characterized in that the nano particle has nucleocapsid, and core is chitosan
The nano combined core of quaternary ammonium salt and insulin composition, shell are hyaluronic acid or sulfhydrylation hyalomitome coated in core surfaces
Acid.
2. nano particle according to claim 1, which is characterized in that the molecular weight of the chitosan quaternary ammonium salt is 50kDa
~200kDa, quaternization degree are 10%~70%；The molecular weight of thiolated hyaluronic acid is 3.4kDa~200kDa, sulfhydrylation journey
Degree is 5%~30%.
3. nano particle according to claim 1, which is characterized in that the grain size of the nano particle is 50~200nm.
4. application of the nano particle as claimed in claim 1 or 2 in preparing Macrulin.
5. a kind of oral insulin medicament preparation, which is characterized in that load pancreas islet comprising claims 1 to 3 any one of them
The nano particle of element.
6. a kind of preparation method of load insulin nano particle, which is characterized in that include the following steps：
S1. by chitosan quaternary ammonium salting liquid by channel 1 and 2, insulin solutions are reached in vortex mixing region and are mixed by 3 and 4
It closes, obtains the nano combined core of insulin and chitosan quaternary ammonium salt composition；Four channel liquids at the uniform velocity flow, flow velocity 2mL/
Min~50mL/min；
S2. by mixed liquor obtained by S1, by channel 1 and 2, hyaluronic acid or thiolated hyaluronic acid solution are arrived by channel 3 and 4
Up to being mixed in vortex mixing region, surface coated with hyaluronic acid or thiolated hyaluronic acid nano particle are obtained；Four channel liquid
Body at the uniform velocity flows, and flow velocity is 2mL/min~50mL/min.
7. preparation method according to claim 6, which is characterized in that the quaternization degree of the chitosan quaternary ammonium salt is
10%~70%；The sulfhydrylation degree of the thiolated hyaluronic acid is 5%~30%.
8. preparation method according to claim 6, which is characterized in that a concentration of the 0.2 of the chitosan quaternary ammonium salt~
3mg/mL, a concentration of 0.5~3mg/mL of insulin solutions.
9. preparation method according to claim 6, which is characterized in that the hyaluronic acid or thiolated hyaluronic acid it is dense
Degree is 0.25~1mg/mL.
10. preparation method according to claim 6, which is characterized in that the quality of the insulin and chitosan quaternary ammonium salt
Than being 0.5~2.
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| PCT/CN2018/101176 WO2019148810A1 (en) | 2018-02-02 | 2018-08-17 | Nanoparticle formulation for oral insulin delivery, and preparation method therefor |
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| WO2019148810A1 (en) * | 2018-02-02 | 2019-08-08 | 中山大学 | Nanoparticle formulation for oral insulin delivery, and preparation method therefor |
| CN110974778A (en) * | 2019-05-14 | 2020-04-10 | 暨南大学 | High-drug-loading-rate slow-release microgel ointment and preparation method and application thereof |
| CN112842929A (en) * | 2019-11-27 | 2021-05-28 | 华熙生物科技股份有限公司 | Sulfhydrylation hyaluronic acid and preparation method and application thereof |
| CN113304124A (en) * | 2021-06-07 | 2021-08-27 | 合肥工业大学 | Oral insulin chitosan nanoparticle solution and preparation method thereof |
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| CN112842929A (en) * | 2019-11-27 | 2021-05-28 | 华熙生物科技股份有限公司 | Sulfhydrylation hyaluronic acid and preparation method and application thereof |
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Application publication date: 20180807 |