CN107412201A - A kind of glucose responding Exenatide microneedle patch and preparation method thereof - Google Patents
A kind of glucose responding Exenatide microneedle patch and preparation method thereof Download PDFInfo
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- CN107412201A CN107412201A CN201710362155.3A CN201710362155A CN107412201A CN 107412201 A CN107412201 A CN 107412201A CN 201710362155 A CN201710362155 A CN 201710362155A CN 107412201 A CN107412201 A CN 107412201A
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- exenatide
- glucose oxidase
- mineralising
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- 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
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
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- 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
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- 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
- A61K47/02—Inorganic compounds
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- 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
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- 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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- 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
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
Abstract
The present invention provides a kind of long-acting microneedle patch of Exenatide, it includes hydrogel matrix, the Exenatide formulation of separate biomimetic mineralization and the glucose oxidase preparation of biomimetic mineralization are loaded with skeleton simultaneously, the Exenatide formulation of the biomimetic mineralization be based in acid condition can be constructed by the bionical mineral of natural degradation mineralized particles, and the glucose oxidase preparation of the biomimetic mineralization is the mineralized particles constructed by based on bionical mineral non-degradable in acid condition；The mass ratio of Exenatide and glucose oxidase is 03 in the skeleton:0‑5；And the content of Exenatide and glucose oxidase is not 0 in skeleton.The long-acting microneedle patch of Exenatide of the present invention is simple, safety, has glucose responding, realizes the long-acting intelligence release of Exenatide, can be efficiently applied to the treatment of type II diabetes.The present invention method that also offer prepares the long-acting microneedle patch of Exenatide.
Description
Technical field
The present invention relates to biological medicine technology, drug controlled release technical field, specifically a kind of novel glucose
Response Exenatide microneedle patch and preparation method thereof.
Background technology
Exendin (exendin-4) be by Southwestern United Stares Monster (Gila monster,
Helodermasuspectum) a kind of caused by salivary gland and natural glucagon-like-peptide-1 (glucagon-like
Peptide-1, GLP-1) similar peptide material.Exenatide (exenatide) is artificial synthesized Exendin, its by
39 amino acid compositions, sequence are as follows：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-
Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-
Pro-Pro-Ser-NH2。
Due to having 53% sequence homology with the GLP-1 of mammal, Exenatide is for GLP-1 by physical performance
Go out high affinity, simultaneously because the difference of sequence, it can effectively evade internal DPP IV
(dipetidylpeptidase IV (DPP IV)) degrades, and shows higher stability.
Pharmacological action mainly includes known to Exenatide：(1) promote pancreatic beta cell glucose responding, stimulate its
Increase insulin secretion when blood glucose is higher, insulin secretion is reduced when blood glucose is normal；(2) abnormal hyperglycemic factor is suppressed
Secretion, glucagon suppression is secreted such as in patients with NIDDM hyperglycaemia environment；(3) rate of evacuation of stomach is slowed down；(4)
Reduce appetite；(5) patients with NIDDM is increased for sensitiveness of insulin etc..These effects show Exenatide in glycosuria
There is great potential in disease treatment (especially type II diabetes).Therefore, Exenatide injection obtains the U.S. in April, 2005
FDA's approval listing is used to treat type II diabetes, trade name Byetta.However, its circulation time in vivo
Too short, half-life period is 60-90 minutes, so its administering mode is twice a day hypodermic injection (60 minutes before breakfast and supper).
High administration frequency reduces the compliance of patient, affects the treatment, and increases cost.Therefore, a kind of convenient, glucose responding of development
, the controlled release that the delivery system of feedback regulation is used for Exenatide there is very high application prospect.
At present, the delivery system of glucose responding consists predominantly of two kinds of modules：Glucose responding module and medicine are released
Amplification module.Wherein the glucose signals of various concentrations mainly can be converted into specific biology by glucose responding module by one kind
The material composition of chemical signal, such as glucose oxidase (glucose oxidase, GOx) are frequently used to oxidizing glucose
Molecule, induce a kind of low ph value, high hydrogen peroxide content, the microenvironment of hypoxemia, the structural change of stimulating drug release module or drop
Solution is to discharge supported medicine.It is pointed out that current glucose responding delivery system is by glucose responding mould
Block and insoluble drug release module are attached in same preparation, and its shortcoming is：Two kinds of components discharge simultaneously, it is impossible to provide long-acting grape
The medicine controlled releasing of sugar response.In addition, current glucose responding system is mostly directly to be subcutaneously injected, easily pain is brought to patient
Hardship, while easily trigger immune and inflammatory reaction, it is unfavorable for the compliance and curative effect of patient.
The content of the invention
In order to overcome the shortcomings of existing Exenatide circulation time is short, frequency of injection is high, it is an object of the invention to provide
A kind of simple, Exenatide microneedle patch of safety, glucose responding, to realize that the long-acting intelligence of Exenatide discharges, make
It can be efficiently applied to the treatment of type II diabetes.
In addition, the present invention also provides a kind of preparation method of the long-acting microneedle patch of Exenatide.
Technical scheme is as follows：
First, there is provided a kind of long-acting microneedle patch of Exenatide, comprising hydrogel matrix, be loaded with simultaneously mutually on skeleton
The Exenatide formulation of independent biomimetic mineralization and the glucose oxidase preparation of biomimetic mineralization, wherein, the biomimetic mineralization
Exenatide formulation be based in acid condition can be constructed by the bionical mineral of natural degradation mineralized particles, it is and described bionical
The glucose oxidase preparation of mineralising is the mineralized particles constructed by based on bionical mineral non-degradable in acid condition；Institute
It is 0-3 to state the mass ratio of Exenatide and glucose oxidase in skeleton:0-5, preferably 3:5；And Exenatide and Portugal in skeleton
The content of grape carbohydrate oxidase is not 0.
In the solution of the present invention, the Exenatide formulation of described biomimetic mineralization can be based on calcium phosphate, calcium carbonate, phosphorus
The Exenatide particle of biomimetic mineralization constructed by any one mineral such as sour magnesium, the Exenatide particle of these biomimetic mineralizations is all
It can be prepared by existing method.In preferred embodiments of the present invention, the Exenatide formulation of described biomimetic mineralization be by
The organic-inorganic composition granule that Exenatide and bionical mineral calcium phosphate are formed, wherein, by weight percentage, Ai Saina
Peptide accounts for the 15~20% of whole particle, and calcium phosphate accounts for the 75~78% of whole particle, and surplus is the crystallization water；The composite particles can
Reaction induced generation using biomimetic mineralization method in situ by acidic amino acid residue on Exenatide and calcium ion.
In the solution of the present invention, the glucose oxidase preparation of described biomimetic mineralization can be based on cupric phosphate, dioxy
The glucose oxidase particle of biomimetic mineralization constructed by any one mineral such as SiClx, iron oxide.The Portugal of these biomimetic mineralizations
Grape carbohydrate oxidase particle can be prepared by existing method.In preferred embodiments of the present invention, described biomimetic mineralization
Glucose oxidase particle is the organic-inorganic composition granule being made up of glucose oxidase and bionical mineral cupric phosphate, its
In, by weight percentage, glucose oxidase accounts for the 12~18% of whole particle, cupric phosphate account for whole particle 72~
76%, surplus is the crystallization water；The composite particles can pass through glucose oxidase upper amino acid using biomimetic mineralization method in situ
The reaction induced generation of amino group and copper ion in residue.
In the solution of the present invention, described hydrophilic gel, which can be selected from, existing a variety of can be used as drug controlled-release body pastern bone
The hydrophilic gel material of frame, including hyaluronic acid, hydroxypropyl methylcellulose, polyethylene glycol oxide, sodium alginate or xanthans etc.；This hair
In bright preferable scheme, described hydrophilic gel material is sodium alginate or hyaluronic acid.
In a kind of embodiment of the present invention, the long-acting microneedle patch of described Exenatide is with based on sodium alginate
Hydrogel is skeleton, and skeleton is loaded with：Mineralising Exenatide particle based on calcium phosphate and the mineralising grape glycosyloxy based on cupric phosphate
Change enzyme granulate, the Exenatide content that every paster is loaded with can regulate and control between 0-300 micrograms, the grape glycosyloxy that every paster is loaded with
Changing enzyme content can regulate and control between 0-500 micrograms.The described mineralising Exenatide particle based on calcium phosphate, the quality of its component
Percentage is：Exenatide is about 18%, and calcium phosphate is about 76%, and the crystallization water is about 6%；The described mineralising based on cupric phosphate
Glucose oxidase particle, the mass percent of its component are：Glucose oxidase is about 15%, and cupric phosphate is about 75%, knot
Brilliant water is about 10%.
In addition, the present invention also provides a kind of preparation method of the long-acting microneedle patch of Exenatide, comprise the following steps：
1) Exenatide and calcic inorganic salts are in cell culture medium, containing CO2Environment in mineralising reaction, be based on
The mineralising Exenatide particle of calcium phosphate；
2) glucose oxidase and cupric inorganic salts are in phosphate buffer, the mineralising reaction in the environment less than room temperature,
Obtain the mineralising glucose oxidase particle based on cupric phosphate；
3) by the mineralising Exenatide particle that step 1) obtains and the mineralising glucose oxidase particle that step 2) obtains, divide
It is not loaded into the microneedle patch skeleton of hydrophilic gel material preparation, makes the quality of Exenatide and glucose oxidase therein
Than for 0-3:0-5.
In a kind of preferred embodiment of the present invention, the preparation method of the long-acting microneedle patch of described Exenatide, including
Following steps：
1. the preparation of mineralising Exenatide particle：Weigh Exenatide to be directly dissolved in cell culture medium, Exenatide
Concentration after dissolving is 1~5 mg/ml；Calcic inorganic salts are added into system, the concentration of calcic inorganic salts is 5 after addition
~15 mM/ls；Containing CO2Environment in mineralising reaction 2~24 hours；Obtain the mineralising Exenatide based on calcium phosphate
Grain；
2. the preparation of mineralising glucose oxidase：Weigh glucose oxidase to be dissolved in phosphate buffer (PBS), grape
Concentration after carbohydrate oxidase dissolving is 0.5~1 mg/ml；Cupric inorganic salts are subsequently to added into reaction system, are contained after addition
The concentration of inorganic copper salt is 0.5~1 mM/l, mineralising reaction 72~96 hours in the environment less than room temperature；It is based on
The mineralising glucose oxidase particle of cupric phosphate；
3. the preparation for the microneedle patch that dual mineralized particles support：2. mineralising glucose oxidase particle that step is obtained
And 1. mineralising Exenatide particle that step obtains is dispersed in PBS and DMEM respectively, two kinds of solution after scattered are distinguished
The cavity surface of the mould of microneedle patch shaping is coated on, it is 0-3 to make the mass ratio of Exenatide and glucose oxidase:0-
5；Sodium alginate soln is added in mold cavity again and brings it about gel, it is long-acting micro- to obtain a kind of Exenatide after drying
Pin paster.
In the above-mentioned preparation scheme of the present invention, described cell culture medium is including but not limited to DMEM in high glucose and low sugar DMEM
Cell culture medium；Described calcic inorganic salts are including but not limited to calcium chloride, calcium nitrate；Described cupric inorganic salts include but not
It is limited to copper chloride, copper sulphate, copper nitrate.
Preferably, step 3. described in coating be by several times be coated with；Every time by the solution coating described in 40~60 microlitres in
Behind described mold cavity surface, first mould is placed under vacuum environment and acted on, reuse plate centrifuge centrifugation；So operation
Repeatedly circulation.
It is further preferred that the pressure of described vacuum environment is 400~500 millimetress of mercury；Described flat board centrifugation
Speed and time are 2000~2500 revs/min, 20~30 minutes.
Preferably, step 3. described in make sodium alginate soln occur gel method be in the sodium alginate soln
Middle addition calcium salt soln, preferably calcium chloride solution.
It is further preferred that described sodium alginate concentration is 2%~3%；Concentration after the calcium chloride of addition for 50~
100 mM/ls.
Preferably, step 3. described in drying time be 24~48 hours.
The long-acting microneedle patch of Exenatide of the present invention is the long-acting microneedle patch for having glucose responding, is passed through
The paster skeleton of hydrophilic gel material preparation will be loaded on after Exenatide and glucose oxidase respectively preparation, realize and pass medicine
The separation of glucose responding module and insoluble drug release module in system.Due to the generation of glucose oxidation reaction, under local ph
Drop, degradable biomimetic mineralization Exenatide pellet degradation under acid condition, discharge Exenatide；Non-degradable under acid condition
Biomimetic mineralization glucose oxidase particle keep stable, ensure long-term glucose responding characteristic.Such a binding mode avoids
Two kinds of module adverse effects that release band is come simultaneously, while biomimetic mineralization enables Exenatide and glucose oxidase efficiently to keep
Bioactivity, the controlled release of Exenatide glucose responding is thus significantly improved, realize the long-acting intelligence release of Exenatide.
Compared with prior art, the application of microneedle patch of the invention will significantly reduce type 2 diabetes patient's administration frequency, while greatly
The big compliance for improving patient's percutaneous dosing.
Brief description of the drawings
Fig. 1 is the transmission electron microscope photo of mineralising Exenatide in embodiment 1.
Fig. 2 is the stereoscan photograph of mineralising glucose oxidase in embodiment 2.
Fig. 3 embodies the glucose responding characteristic of dual mineralized particles system in embodiment 3.
Fig. 4 is the stereoscan photograph of microneedle patch prepared by embodiment 4.
Fig. 5 is the mineralising Exenatide and mineralising glucose oxidase that are mounted with fluorescence labeling prepared by embodiment 4
Fluorescence photo.
Fig. 6 embodies Regulation of blood glucose effect inside described in embodiment 5 and compared.
Embodiment
The present invention is specifically described below by embodiment, the present embodiment is served only for making further the present invention
It is bright, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art makes according to the content of foregoing invention
Some nonessential modifications and adaptations, belong to the scope of the present invention.
The preparation of the mineralising Exenatide particle of embodiment 1.
Weigh 2 milligrams of Exenatides to be dissolved in 1 milliliter of DMEM cell culture fluid, the solution is put in 37 degrees Celsius of environment
Put 24 hours.Weigh 55 milligrams of calcium chloride solids to be dissolved in 0.5 milliliter of ultra-pure water, obtain 1 mol/L calcium chloride solution.It is micro- by 10
Rise calcium chloride solution to be added in 1 milliliter of Exenatide DMEM solution, react 24 in 37 degrees Celsius, 5% carbon dioxide environment
Hour.Resulting solution is centrifuged by ultrafiltration, molecular cut off 100k, and centrifugal speed is 6000 revs/min, and centrifugation time 20 divides
Clock, obtains mineralising Exenatide particle, the pattern arrived using transmission electron microscope observation, as shown in Figure 1.
The preparation of the mineralising glucose oxidase particle of embodiment 2.
Weigh 3 milligrams of glucose oxidases to be dissolved in 6 milliliters of PBS, after 0.5 hour, 40 microlitres are added into solution
Copper sulphate (120 mM/ls) solution.Solution is placed under 4 degrees Celsius of environment and reacted three days.Obtained blue precipitate centrifugation
Separate (6000 revs/min, centrifugation time 10 minutes), obtain mineralising glucose oxidase particle, use SEM
Its pattern is observed, as shown in Figure 2.
Drug release in vitro of the embodiment 3. based on concentration of glucose
It is micro- that prepared by the mineralized particles for being loaded with 300 microgram Exenatides and embodiment 2 prepared by embodiment 1 is loaded with 500
The mineralized particles of gram glucose oxidase, are distributed in 300 microlitre 2% of sodium alginate, and 15 microlitres are then added into system
Calcium chloride (1 mol/L) solution, obtain being loaded with the hydrogel of dual mineralized particles.Hereafter, hydrogel is inserted into various concentrations
In the glucose solution of (0,1,4 mg/ml).In different time points, 50 microlitres of supernatants are taken out, utilize the Ai Sai of commercialization
That peptide enzyme linked immunological kit determines the content of Exenatide.As a result (as shown in Figure 3) is shown, (4 millis under hyperglycaemia environment
Grams per milliliter) Exenatide quick release (curve of Fig. 3 intermediate cams shape mark), and in euglycemia environment (1 mg/ml)
Or in control group (0 mg/ml), a small amount of medicine is released (curve marked respectively by round dot and square in Fig. 3), it was demonstrated that
The glucose responding characteristic of dual mineralized particles system proposed by the present invention.
The preparation for the sodium alginate microneedle patch that 4. dual mineralized particles of embodiment support
The mineralising glucose oxidase particle prepared according to the method for embodiment 2 is dispersed in 300 microlitres of PBS first, so
Afterwards according to below scheme circulate operation six times：50 microlitres of dispersion liquids are taken to be coated in the hole table of the silica gel mould of microneedle patch shaping
Face, mould is placed in the vacuum environment of 400 millimetress of mercury after acting on 10 minutes, mould is gone in six orifice plates, flat board from
(2500 revs/min, 20 minutes) are centrifuged in scheming.
After completing above-mentioned circulate operation, it is micro- that the mineralising Exenatide particle prepared according to the method for embodiment 1 is dispersed in 300
Rise in DMEM, the above-mentioned coating of same repetitive cycling, application of vacuum, centrifugation etc. operate six times.
Hereafter, 0.75 × 4 inch of adhesive tape is sticked in silica gel mould surrounding side wall, is then added to die surface
3 milliliter 2% of sodium alginate soln, add 150 microlitres of calcium chloride solutions (1 mol/L).Dried 48 hours in drier, it is small
Heart strips down microneedle patch from mould, 4 degrees Celsius of preservations, obtains glucose responding Exenatide microneedle patch.
It is as shown in Figure 4 with scanning electron microscopic observation microneedle patch, its pattern.
In addition, it is utilized respectively fluorescent dye fluorescein isothiocynate (fluorescein isothiocyanate, FITC)
Exenatide and glucose oxidase are marked with 3H- indoles cyanines types bioluminescence sign dyestuff (Cyanine 5, Cy5), and is utilized
Above method structure supports the microneedle patch of the mineralized particles of fluorescence labeling, uses fluorescence microscope (as shown in Figure 5).
The preparation for the hyaluronic acid microneedle patch that 5. dual mineralized particles of embodiment support
The mineralising glucose oxidase particle prepared according to the method for embodiment 2 is dispersed in 300 microlitres of PBS first, so
Afterwards according to below scheme circulate operation six times：50 microlitres of dispersion liquids are taken to be coated in the hole table of the silica gel mould of microneedle patch shaping
Face, mould is placed in the vacuum environment of 400 millimetress of mercury after acting on 10 minutes, mould is gone in six orifice plates, flat board from
(2500 revs/min, 20 minutes) are centrifuged in scheming.
After completing above-mentioned circulate operation, it is micro- that the mineralising Exenatide particle prepared according to the method for embodiment 1 is dispersed in 300
Rise in DMEM, the above-mentioned coating of same repetitive cycling, application of vacuum, centrifugation etc. operate six times.
Hereafter, 0.75 × 4 inch of adhesive tape is sticked in silica gel mould surrounding side wall, is then added to die surface
3 milliliters of 4wt% hyaluronic acid solution, about 60 milligrams of N, N'- methylene-bisacrylamides are added thereto, while add about 15
Milligram light trigger Irgacure 2959, after drying 48 hours in drier, mould is placed on the ultraviolet of 365 nano wave lengths
Act on 1 minute under light, carefully strip down microneedle patch from mould, 4 degrees Celsius of preservations.
Regulation of blood glucose inside the glucose responding Exenatide microneedle patch of embodiment 6.
9 C57BL/6 diabetic mices (6-8 weeks, male, 40-50 grams of body weight) are randomly divided into three groups, every group three, institute
There is mouse freely to ingest, drink water, 12 hours day-night cycles are set in animal housing, and maintain room temperature in (22 ± 2) degree Celsius.First
Group mouse is (control group) without any processing；Second group of mouse subcutaneous injection Exenatide (20 microgram)；3rd group of mouse is first
Depilation is handled at skin of back, glucose responding Exenatide microneedle patch (Exenatide content prepared by embodiment 4
For 20 micrograms) exposed skin surface is pressed against, fixed using medical adhesive tape.Hereafter, different time points (0,0.25,1,2,3,
4th, 6,12,18,24,36,48,60,72 hours) from tail vein 5 microlitres of blood is taken, blood sugar level is determined using blood glucose meter.As a result demonstrate,prove
Microneedle patch prepared by bright (as shown in Figure 6) embodiment 4 can control blood glucose more for a long time normal relative to common Exenatide
Level, and can effectively extend blood sugar recovery to the time of previous level.
Claims (10)
1. a kind of long-acting microneedle patch of Exenatide, it is loaded with simultaneously comprising hydrogel matrix, on skeleton separate bionical
The Exenatide formulation of mineralising and the glucose oxidase preparation of biomimetic mineralization, wherein, the Exenatide system of the biomimetic mineralization
Agent be based in acid condition can be constructed by the bionical mineral of natural degradation mineralized particles, and the grape of the biomimetic mineralization
Oxidase enzyme preparation is the mineralized particles constructed by based on bionical mineral non-degradable in acid condition；Ended in the skeleton
The mass ratio for filling in that peptide and glucose oxidase is 0-3:0-5, preferably 3:5；And Exenatide and glucose oxidase in skeleton
Content be 0.
2. the long-acting microneedle patch of Exenatide described in claim 1, it is characterised in that：The Exenatide of described biomimetic mineralization
It is the Exenatide particle based on the biomimetic mineralization constructed by any one in calcium phosphate, calcium carbonate or magnesium phosphate.
3. the long-acting microneedle patch of Exenatide described in claim 2, it is characterised in that：Described imitating based on calcium phosphate structure
Rawore Exenatide particle is the organic-inorganic composition granule being made up of Exenatide and bionical mineral calcium phosphate, wherein,
By weight percentage, Exenatide accounts for the 15~20% of whole nano particle, calcium phosphate account for whole nano particle 75~
78%, surplus is the crystallization water.
4. the long-acting microneedle patch of Exenatide described in claim 1, it is characterised in that：The grape glycosyloxy of described biomimetic mineralization
It is the glucose oxidase based on the biomimetic mineralization constructed by any one in cupric phosphate, silica or iron oxide to change enzyme.
5. the long-acting microneedle patch of Exenatide described in claim 4, it is characterised in that：Described imitating based on cupric phosphate structure
Rawore glucose oxidase particle is the organic-inorganic composition being made up of glucose oxidase and bionical mineral cupric phosphate
Grain, wherein, by weight percentage, glucose oxidase accounts for the 12~18% of whole nano particle, and cupric phosphate accounts for whole nanometer
The 72~76% of particle, surplus are the crystallization water.
6. the long-acting microneedle patch of Exenatide described in claim 1, it is characterised in that：Described hydrophilic gel can be selected from saturating
Bright matter acid, hydroxypropyl methylcellulose, polyethylene glycol oxide, sodium alginate or xanthans；It is preferred that sodium alginate or hyaluronic acid.
7. the long-acting microneedle patch of Exenatide described in claim 1, it is characterised in that：Using based on the hydrogel of sodium alginate as
Skeleton, skeleton are loaded with：Mineralising Exenatide particle based on calcium phosphate and the mineralising glucose oxidase particle based on cupric phosphate,
The Exenatide content that every paster is loaded with can regulate and control between 0-300 micrograms, the glucose oxidase content that every paster is loaded with
It can regulate and control between 0-500 micrograms；The described mineralising Exenatide particle based on calcium phosphate, the mass percent of its component
For：Exenatide is 18%, calcium phosphate 76%, the crystallization water 6%；The described mineralising glucose oxidase based on cupric phosphate
Particle, the mass percent of its component are：Glucose oxidase is 15%, calcium phosphate 75%, the crystallization water 10%.
8. a kind of method for preparing the long-acting microneedle patch of Exenatide, comprises the following steps：
1) Exenatide and calcic inorganic salts are in cell culture medium, containing CO2Environment in mineralising reaction, obtain being based on phosphoric acid
The mineralising Exenatide particle of calcium；
2) glucose oxidase the mineralising reaction in the environment less than room temperature, obtains with cupric inorganic salts in phosphate buffer
Mineralising glucose oxidase particle based on cupric phosphate；
3) by the mineralising Exenatide particle that step 1) obtains and the mineralising glucose oxidase particle that step 2) obtains, add respectively
The microneedle patch skeletal internal of hydrophilic gel material preparation is downloaded to, makes the mass ratio of Exenatide and glucose oxidase therein
For 0-3:0-5.
9. the method described in claim 8, comprises the following steps：
1. the preparation of mineralising Exenatide particle：Weigh Exenatide to be directly dissolved in cell culture medium, Exenatide dissolving
Concentration afterwards is 1~5 mg/ml；Calcic inorganic salts are added into system, the concentration of calcic inorganic salts is 5~15 after addition
MM/l；Containing CO2Environment in mineralising reaction 2~24 hours；Obtain the mineralising Exenatide particle based on calcium phosphate；
2. the preparation of mineralising glucose oxidase：Weigh glucose oxidase to be dissolved in phosphate buffer (PBS), grape glycosyloxy
The concentration changed after enzyme dissolving is 0.5~1 mg/ml；Cupric inorganic salts are subsequently to added into reaction system, after addition cupric without
The concentration of machine salt is 0.5~1 mM/l, mineralising reaction 72~96 hours in the environment less than room temperature,；Obtain being based on phosphorus
The mineralising glucose oxidase particle of sour copper；
3. the preparation for the microneedle patch that dual mineralized particles support：2. mineralising glucose oxidase particle that step is obtained and
1. mineralising Exenatide particle that step obtains is dispersed in PBS and DMEM respectively, and two kinds of solution after scattered are respectively coated
In the cavity surface of the mould of microneedle patch shaping, it is 0-3 to make the mass ratio of Exenatide and glucose oxidase:0-5；Again
Sodium alginate soln is added in mold cavity and brings it about gel, obtains the described long-acting micropin of Exenatide after drying
Paster；
The described method for optimizing for making sodium alginate soln that gel occur is to add calcium salt soln in the sodium alginate soln；
Further preferably add calcium chloride solution；It is furthermore preferred that described sodium alginate soln concentration is 2%~3%；The chlorination of addition
Concentration after calcium is 50~100 mM/ls；
Described drying time is preferably 24~48 hours.
10. the method described in claim 9, it is characterised in that：Step 3. described in coating be by several times be coated with；Every time by 40~
Mould with behind described mold cavity surface, being first placed under vacuum environment and acting on, reuse by the solution coating described in 60 microlitres
Plate centrifuge centrifuges；So operation repeatedly circulation；The pressure of described vacuum environment is preferably 400~500 millimetress of mercury；Institute
The speed for the flat board centrifugation stated is preferably 2000~2500 revs/min；Preferably 20~30 minutes time of described flat board centrifugation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710362155.3A CN107412201B (en) | 2017-05-22 | 2017-05-22 | Glucose-responsive exenatide microneedle patch and preparation method thereof |
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| WO2023040568A1 (en) * | 2021-09-14 | 2023-03-23 | 中国科学院理化技术研究所 | Insoluble transdermal microneedle patch, preparation method therefor and application thereof |
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| WO2023040568A1 (en) * | 2021-09-14 | 2023-03-23 | 中国科学院理化技术研究所 | Insoluble transdermal microneedle patch, preparation method therefor and application thereof |
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