CN105770903B - A kind of preparation method of temperature control drug-releasing polymer micro-sphere material - Google Patents
A kind of preparation method of temperature control drug-releasing polymer micro-sphere material Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of temperature control drug-releasing polymer micro-sphere material, steps are as follows: chitosan is dissolved in lactic acid solution, succinic anhydride is added and is reacted to obtain water-soluble succinyl-chitosan；Product is dissolved in buffer solution, and chaff amine -2- furylamine is added dropwise, carbodiimides is then added and is reacted to obtain furans chitosan；Sodium alginate is soluble in water, sodium periodate solution is added dropwise and reacts to obtain aldehyde radical sodium alginate；Aldehyde radical sodium alginate is soluble in water, 4- (4-N- maleimide phenyl) butyric acid reactant salt is added and obtains maleimation sodium alginate；Furans chitosan aqueous solution and maleimation sodium alginate aqueous solution are mixed in isometric ratio, add ultrasonic emulsification in the paraffin oil containing emulsifier Span 80；Emulsion is stirred and is volatilized, pours into and polymer microballoon is precipitated in isopropanol, freeze-drying obtains product after cleaning.The present invention significantly improves the safe handling of micro-sphere material, there is major application value in terms of medicine controlled releasing and gene therapy.
Description
Technical field
The invention belongs to field of biomedical polymer materials, and in particular to one kind is based on Diels-Alder cross-linking reaction
Temperature control drug-releasing polymer micro-sphere material preparation method.
Background technique
Micro-sphere material with biodegradability can be used for the embedding and release of drug, albumen or gene, extensively at present
The general research for fields such as medicine controlled releasing, organizational project and regenerative medicines.Select suitable microballoon system, it can be achieved that drug exists
Intracorporal slow release reaches ideal therapeutic effect.Natural polymer has excellent biocompatibility and adjustable biology
Degradability is the desired matrix material for preparing microballoon.Common polysaccharide includes: chitosan, sodium alginate, hyaluronic acid, fibre
Tie up element etc..
In organizational project and regenerative medicine field, polysaccharide microsphere material is often prepared for embedding cell growth factor and base
Cause increases the tissue inducibility of bracket in vivo to improve the bioactivity of cytoskeleton.Using intelligentized controlled release hand
Section, for example the release of drug is controlled, it can be achieved that the targeted delivery of Porcine HGF in vivo, reaches more by adjusting temperature
Ideal therapeutic effect.But existing polysaccharide microsphere material also cannot achieve Porcine HGF isoreactivity protein drug and exist
Intracorporal temperature control transmitting.In addition, polysaccharide microsphere generally uses chemical cross-linking agent during the preparation process, this is easy to cause embedding
Protein drug is denaturalized and inactivates.Such as it is avoided that the polysaccharide microsphere material for being then expected to obtain high bioactivity using chemical cross-linking agent
Material.
(Chinese patent drug, 2014,36 (3): 620~622) reporting using capsicum alkali extracting solution as raw material document 1, is prepared for peppery
Green pepper alkali-chitosan/sodium alginate micro ball, optimizes preparation process using factor method, and has studied its external slow release effect.Most preferably
Preparation process condition are as follows: sodium alginate mass concentration is 15g/L, calcium chloride dosage is 4 times of capsaicine sodium alginate mixing liquids
Product, chitosan dosage are 2 times of capsaicine sodium alginate mixeding liquid volumes, capsaicine concentrate mass concentration is 1.49g/L, preparation
Capsaicine-chitosan/sodium alginate micro ball encapsulation rate reach 43.6%, drugloading rate 18.2mg/g.
(2012,28 (4): liberation army Acta Pharmaceutica Sinica it is micro- 311~314) to report a kind of sodium alginate-chitosan to document 2
The synthesis of ball and its application in terms of immobilization fibrin ferment are oil phase with the mixed solution of atoleine and Span 80,1.5%
Sodium alginate soln is water phase, and suitable calcium chloride is added in chitosan acetic acid (1%) solution as crosslinking agent, is handed over using emulsification
Connection method prepares microballoon.Water phase is added dropwise in oily phase, O/W type emulsion is stirred into, crosslinking agent is added drop-wise in stirring shape
In the emulsion of state, drop Bi Jixu stirs 30min, and centrifugation discards upper oil phase, petroleum ether lower layer microballoon twice after, freezing is dry
It is dry.The microballoon has the ability of certain absorption fibrin ferment as the result is shown.
(2007,23 (1): polymer material science and engineering it is micro- 189~191) to report a kind of chitosan magnetic to document 3
Grain material is used to load the research of 5 FU 5 fluorouracil, using crosslinking-polymerization, by ferriferrous oxide particles, 5 FU 5 fluorouracil and
The acetum of 100mL chitosan is vigorously stirred together to be mixed them thoroughly, and then under the action of ultrasonic wave, is slowly dropped to
In the olive oil and anionic dispersing agents of 80mL, the glutaraldehyde solution that concentration 50% is then added is crosslinking agent, 60 DEG C~90
It is reacted 1~2 hour under the conditions of DEG C, has obtained the nanosphere with magnetic induction performance, measuring its drugloading rate is 21.3%, drug
Encapsulation rate is 55.4%, and the cumulative release rate in 30 hours is 67.6%.
(2011,7 (8): Acta Biomaterialia 3050~3059) reports a kind of hyaluronic acid-heparin to document 4
Polysaccharide composite microsphere material is used to deliver the research of Porcine HGF, using reversed emulsion polymerization, by hyaluronic acid and
Heparin is dissolved in the sodium hydroxide solution that concentration is 0.5M respectively, is separately added into emulsifier and acutely shakes, then mixes the two,
Continue that lower addition chemical cross-linking agent divinylsulfone fullys shake, is vigorously stirred 1 hour, finally pours into reaction solution at room temperature
Polysaccharide microsphere is precipitated in acetone, measuring its average grain diameter is 1 microns, and it is 186.6ng/mg that highest, which carries concentration,.
The preparation method of above-mentioned polysaccharide microsphere material has the following deficiencies:
(1) microballoon containing sodium alginate mostly uses calcium chloride to be crosslinked.Since in physiological conditions, calcium ion can not be kept away
Exempt to replace with other divalent ions, this causes microballoon easily to occur to dissolve and lose slow-release capability.
(2) polysaccharide microsphere prepared by achievees the purpose that discharge drug, this sustained release by the degradation of material itself
It can not achieve effective control, it is difficult to which the purpose for realizing intelligence transmitting drug causes administering effect poor.
(3) microballoon also is crosslinked using chemical reagent such as glutaraldehyde or divinylsulfones, these chemical cross-linking agents tool
There is cytotoxicity, cross-linking process leads to albuminous degeneration, and toxic residue will also generate toxic side effect, it is difficult to carry out clinical use.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of temperature control drug-releasing polymer micro-sphere material, use anti-
Phase emulsion crosslinking technological makes microballoon generate temperature sensitivity by Diels-Alder reaction come crosslinked microsphere.
The technical solution for realizing the aim of the invention is as follows:
A kind of preparation method of temperature control drug-releasing polymer micro-sphere material, comprising the following steps:
Chitosan is dissolved in lactic acid solution at room temperature by step 1, and succinic anhydride is added, is reacted, will be reacted molten at room temperature
Water-soluble succinyl-chitosan is obtained after liquid dialysis freeze-drying；
Succinyl-chitosan is dissolved in buffer solution by step 2 at room temperature, and chaff amine -2- furans first is added dropwise into solution
Then amine is added carbodiimides and is reacted, obtains furans chitosan after dialysis freeze-drying；
It is step 3, at room temperature that sodium alginate is soluble in water, sodium periodate solution is added dropwise under the conditions of being protected from light, after reaction thoroughly
It analyses and is lyophilized to obtain aldehyde radical sodium alginate；
Step 4, aldehyde radical sodium alginate is soluble in water, addition 4- (4-N- maleimide phenyl) butyric acid reactant salt,
Maleimation sodium alginate is obtained after dialysis freeze-drying；
Step 5 prepares furans chitosan aqueous solution and maleimation sodium alginate aqueous solution respectively, at room temperature will
Prepared two kinds of solution is sufficiently mixed in isometric ratio, then the stone containing emulsifier Span 80 is added in mixed solution
In wax oil, ultrasonic emulsification obtains microballoon emulsion；
Step 6 stays overnight emulsion stirring volatilization, then pours into lotion and polymer microballoon is precipitated in isopropanol, then
It is successively cleaned respectively with isopropanol, n-hexane and acetone, finally freeze-drying obtains temperature control drug-releasing polymer microballoon
Material.
Compared with prior art, the present invention its remarkable advantage is: (1) crosslinking of polymer microballoon is based on Diels-
Alder reaction, does not chemically react with the active medicine that is embedded, the bioactivity of energy effective protection drug, especially suitable for
The embedding of protein medicaments；(2) it avoids having used toxic chemical crosslinking agent in preparation, therefore non-toxic reagent residual in microballoon,
Both the natural activity for having remained polysaccharide in turn ensures the safety in utilization of micro-sphere material；(3) present invention process and equipment letter
Single, easy, safe operation has many advantages, such as that preparation temperature is low, curing rate is fast, process cycle is short, does not generate pollution to environment,
It is suitble to commercially produce.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of temperature control drug-releasing polymer microballoon prepared by the method for the present invention.
Fig. 2 is the scanning electron figure of microballoon under different temperatures.
Fig. 3 is the graph of relation of microsphere diameter and temperature change.
Fig. 4 is the graph of relation of microspheres swell rate and temperature change.
Fig. 5 is weight-loss curve figure of the microballoon at 37 DEG C in phosphate buffer solution (PBS).
Fig. 6 is the drug release behavior curve graph for changing microballoon under temperature conditions.
Specific embodiment
With reference to the accompanying drawing and specific embodiment the present invention will be further described.
Raw materials and reagents: chitosan, sodium alginate, sodium metaperiodate, chaff amine -2- furylamine, water-soluble carbodiimide
(EDC) it is purchased from Sigma Co., USA；Succinic anhydride analyzes pure, Chinese Medicine group；Lactic acid analyzes pure, Solution on Chemical Reagents in Shanghai
Co., Ltd；4- (4-N- maleimide phenyl) butyrate (MPBH), is purchased from Thermo Fisher company of the U.S..Span 80,
Analyze pure, Nanjing Chemistry Reagent Co., Ltd.；Isopropanol, n-hexane, acetone analyze pure, Solution on Chemical Reagents in Shanghai Co., Ltd；Chlorine
Change sodium, potassium chloride, analyzes pure, three factory of Shanghai reagent；Disodium hydrogen phosphate, potassium dihydrogen phosphate, analysis is pure, and Hangzhou chemical reagent is limited
Company.Bone morphogenetic protein (BMP-2) and enzyme linked immunosorbent assay (ELISA) (ELISA) kit, are purchased from U.S. R&D Systems
Company.
The present invention uses ANOVA method of analysis of variance, and significant difference value p is set as≤0.05.
The preparation method of temperature control drug-releasing polymer micro-sphere material of the present invention, specifically includes the following steps:
0.5 gram of chitosan is dissolved in the lactic acid solution that 40 milliliters of concentration are 5% (v/v) at room temperature by step 1, and stirring is 3 small
When form homogeneous solution, the succinic anhydride of 1~4 times of chitosan mass is then added, stir at room temperature carry out within 12~24 hours it is anti-
It answers, water-soluble succinyl-chitosan will be obtained after reaction solution dialysis freeze-drying；
0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of phosphate buffer solutions by step 2 at room temperature, to solution
The middle chaff amine -2- furylamine for being added dropwise 100~500 microlitres, is then added 1/5~1/2 times of succinyl-chitosan quality
It stirs 12~24 hours and is reacted at room temperature after carbodiimides, obtain furans chitosan after dialysis freeze-drying；
1.0 grams of sodium alginate is dissolved in 100 milliliters of water by step 3 at room temperature, and 2~5 milliliters are added dropwise under the conditions of being protected from light
Concentration is the sodium periodate solution of 0.5M, is stirred 1~4 hour under the conditions of being protected from light, and dialyses after reaction and is lyophilized to obtain aldehyde radicalization sea
Mosanom；
1.0 grams of aldehyde radical sodium alginate is dissolved in 100 milliliters of water by step 4, and it is 0.5% that 3~12 milliliters of concentration, which are added,
4- (4-N- maleimide phenyl) butyric acid reactant salt, at room temperature react 0.5~2 hour, dialysis freeze-drying after obtain Malaysia acyl
Imidization sodium alginate；
Step 5 prepares furans chitosan aqueous solution and maleimation sodium alginate aqueous solution, the furan of preparation respectively
The concentration of muttering chitosan aqueous solution and maleimation sodium alginate aqueous solution is 0.5%~2.0% (w/v), room temperature
It is lower to be sufficiently mixed prepared two kinds of solution in isometric ratio, then mixed solution is added and contains emulsifier Span 80
Paraffin oil in, the volume of the emulsifier Span 80 is the 1/20~1/10 of paraffin oil, is obtained within ultrasonic emulsification 2~5 minutes micro-
Ball emulsion；
Step 6 stays overnight emulsion stirring volatilization, then pours into lotion and polymer microballoon is precipitated in isopropanol, then
It is successively cleaned respectively with isopropanol, n-hexane and acetone, finally freeze-drying obtains temperature control drug-releasing polymer microballoon
Material.
The pattern and droplet measurement of microballoon: by the microballoon metal spraying (Cressington 108Auto) after drying, in scanning electricity
Microscopic appearance and analysis partial size are observed on sub- microscope (FEI SIRION100).
The weightless detection of microballoon: certain dry microspheres weighing (W is taken0), the phosphate buffer solution (PBS) being placed at 37 DEG C
Middle incubation.When test, microballoon is centrifuged from solution, is respectively washed with distilled water and acetone, weigh (W after vacuum dryingt), often
A sample parallel testing 5 times.The formula for calculating nanosphere weightlessness is 100% × (W0–Wt)/W0, nanosphere retain quality be
1-100% × (W0–Wt)/W0.The preparation method of phosphate buffer are as follows: weigh analysis 8 grams of pure sodium chloride, 0.2 gram of potassium chloride,
2.9 grams of disodium hydrogen phosphate, 0.2 gram of potassium dihydrogen phosphate, are dissolved in 1000 milliliters of distilled water.
The load and release of drug: being dissolved in phosphate buffer for bone morphogenetic protein, and obtaining concentration is 10 micrograms/milli
The solution risen, then dry polymer microballoon is placed in the 37 DEG C solution and is incubated for 2 hours, then microballoon is taken out in centrifugation, uses
Phosphate buffer cleans centrifugation three times repeatedly.When carrying out release test, the microballoon of adhesion protein drug is placed in 37 DEG C of phosphorus
In phthalate buffer, phosphate buffer is drawn in different time points, according to enzyme linked immunosorbent assay (ELISA) (ELISA) kit mark
Quasi- operating process executes quantitative analysis.
Further detailed description is done to the present invention below with reference to embodiment, but these examples are not intended to limit this hair
It is bright.
Embodiment 1:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
2 grams of succinic anhydrides are then added in even solution, stir 24 hours at room temperature, reaction solution dialysis freeze-drying is then obtained succinyl
Chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 500 microlitres
Chaff amine -2- furylamine stirs 24 hours at room temperature after 0.25 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugared (structural schematic diagram is shown in Fig. 1)；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, the height that 5 milliliters of concentration are 0.5M is then added dropwise
Sodium iodide solution stirs 2 hours under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 10 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 2 hours at room temperature, and dialysis freeze-drying obtains maleimide
Amination sodium alginate (structural schematic diagram is shown in Fig. 1)；
(5) the furans chitosan and maleimation sodium alginate that compound concentration is 0.5% (w/v) respectively are water-soluble
Liquid at room temperature by being sufficiently mixed in equal volume, then is added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein Span
4 milliliters of 80 volume, ultrasonic emulsification 5 minutes；
(6) emulsion for obtaining step 4 is stayed overnight at 40 DEG C with 1000rpm stirring volatilization, and lotion is then poured into isopropyl
Microballoon is precipitated in alcohol, is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.The pattern of resulting polymers microballoon
See Fig. 2, balling-up is good；The diameter of microballoon becomes larger under higher temperature, shows temperature-responsive.
Fig. 3 is the relationship of microsphere diameter and temperature change, the results showed that the diameter of microballoon increases with the raising of temperature, than
Such as, it is 1.01 microns that the average grain diameter of microballoon, which is the average grain diameter of microballoon at 0.72 micron, 45 DEG C, at 37 DEG C, and microballoon at 60 DEG C
Average grain diameter increase to 1.23 microns.
Fig. 4 is the relationship of microspheres swell rate and temperature change, shows that the swelling ratio of microballoon also increases with increasing temperature,
It is consistent with the result that microsphere diameter varies with temperature.
Fig. 5 is weightlessness of the microballoon at 37 DEG C in phosphate buffer solution (PBS), shows that the microballoon has biodegrade
Property, and degradation speed is more stable.
Fig. 6 is that bone morphogenetic protein is from the release in microballoon under change temperature conditions, as the result is shown at relatively high temperatures
(45 DEG C) microballoon occurs swelling and rate of release is caused to increase, and shows that the material is that have preferable temperature control Release Performance.
Embodiment 2:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
1.5 grams of succinic anhydrides are then added in even solution, stir 20 hours at room temperature, reaction solution dialysis freeze-drying is then obtained amber
Acyl chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 400 microlitres
Chaff amine -2- furylamine stirs 20 hours at room temperature after 0.2 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugar；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, it is 0.5M's that 4.5 milliliters of concentration, which are then added dropwise,
Sodium periodate solution stirs 3 hours under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 12 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 1.8 hours at room temperature, and dialysis freeze-drying obtains Malaysia acyl
Imidization sodium alginate；
(5) the furans chitosan and maleimation sodium alginate that compound concentration is 0.6% (w/v) respectively are water-soluble
Liquid at room temperature by being sufficiently mixed in equal volume, then is added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein Span
3 milliliters of 80 volume, ultrasonic emulsification 4 minutes；
(6) emulsion is stayed overnight at 40 DEG C with 800rpm stirring volatilization, then lotion is poured into, microballoon is precipitated in isopropanol,
It is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.
Embodiment 3:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
1.0 grams of succinic anhydrides are then added in even solution, stir 16 hours at room temperature, reaction solution dialysis freeze-drying is then obtained amber
Acyl chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 350 microlitres
Chaff amine -2- furylamine stirs 18 hours at room temperature after 0.15 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugar；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, the height that 4 milliliters of concentration are 0.5M is then added dropwise
Sodium iodide solution stirs 4 hours under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 8 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 1.5 hours at room temperature, and dialysis freeze-drying obtains Malaysia acyl
Imidization sodium alginate；
(5) the furans chitosan and maleimation sodium alginate aqueous solution that compound concentration is 0.8% respectively, room temperature
Under by being sufficiently mixed in equal volume, then be added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein the body of Span 80
3 milliliters, ultrasonic emulsification 4 minutes of product；
(6) emulsion is stayed overnight at 40 DEG C with 900rpm stirring volatilization, then lotion is poured into, microballoon is precipitated in isopropanol,
It is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.
Embodiment 4:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
0.5 gram of succinic anhydride is then added in even solution, stirs 12 hours at room temperature, reaction solution dialysis freeze-drying is then obtained amber
Acyl chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 300 microlitres
Chaff amine -2- furylamine stirs 15 hours at room temperature after 0.12 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugar；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, it is 0.5M's that 3.5 milliliters of concentration, which are then added dropwise,
Sodium periodate solution stirs 1 hour under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 5 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 1.5 hours at room temperature, and dialysis freeze-drying obtains Malaysia acyl
Imidization sodium alginate；
(5) the furans chitosan and maleimation sodium alginate that compound concentration is 1.0% (w/v) respectively are water-soluble
Liquid at room temperature by being sufficiently mixed in equal volume, then is added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein Span
3 milliliters of 80 volume, ultrasonic emulsification 3 minutes；
(6) by emulsion 40 DEG C with 1100rpm stirring volatilization overnight, then lotion is poured into isopropanol be precipitated it is micro-
Ball is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.
Embodiment 5:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
1.5 grams of succinic anhydrides are then added in even solution, stir 14 hours at room temperature, reaction solution dialysis freeze-drying is then obtained amber
Acyl chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 200 microlitres
Chaff amine -2- furylamine stirs 12 hours at room temperature after 0.1 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugar；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, the height that 3 milliliters of concentration are 0.5M is then added dropwise
Sodium iodide solution stirs 2 hours under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 3 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 1 hour at room temperature, and dialysis freeze-drying obtains maleimide
Amination sodium alginate；
(5) the furans chitosan and maleimation sodium alginate that compound concentration is 1.5% (w/v) respectively are water-soluble
Liquid at room temperature by being sufficiently mixed in equal volume, then is added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein Span
2 milliliters of 80 volume, ultrasonic emulsification 3 minutes；
(6) by emulsion 40 DEG C with 1200rpm stirring volatilization overnight, then lotion is poured into isopropanol be precipitated it is micro-
Ball is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.
Embodiment 6:
(1) 0.5 gram of chitosan is added in the lactic acid solution of 40 milliliter 5% (v/v), stirs 3 hours at room temperature, is formed equal
2.0 grams of succinic anhydrides are then added in even solution, stir 20 hours at room temperature, reaction solution dialysis freeze-drying is then obtained amber
Acyl chitosan；
(2) 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of buffer solutions at room temperature, is then added dropwise 100 microlitres
Chaff amine -2- furylamine stirs 12 hours at room temperature after 0.18 gram of carbodiimides is added, and it is poly- that dialysis freeze-drying obtains furans shell
Sugar；
(3) 1.0 grams of sodium alginates are dissolved in 100 milliliters of water at room temperature, the height that 2 milliliters of concentration are 0.5M is then added dropwise
Sodium iodide solution stirs 3 hours under the conditions of being protected from light, and dialysis freeze-drying obtains aldehyde radical sodium alginate；
(4) 1.0 grams of aldehyde radical sodium alginates are dissolved in 100 milliliters of water at room temperature, 6 milliliters of concentration, which are then added, is
0.5% 4- (4-N- maleimide phenyl) butyric acid salting liquid reacts 0.5 hour at room temperature, and dialysis freeze-drying obtains Malaysia acyl
Imidization sodium alginate；
(5) compound concentration is 2% (w/v) respectively furans chitosan and maleimation sodium alginate aqueous solution,
It at room temperature by being sufficiently mixed in equal volume, then is added in 40 milliliters of paraffin oils containing emulsifier Span 80, wherein Span 80
2 milliliters of volume, ultrasonic emulsification 2 minutes；
(6) by emulsion 40 DEG C with 1500rpm stirring volatilization overnight, then lotion is poured into isopropanol be precipitated it is micro-
Ball is then respectively washed with isopropanol, n-hexane and acetone, is finally lyophilized.
Above embodiments cover most representative experimental data.
In conclusion the preparation method of temperature control drug-releasing polymer micro-sphere material of the present invention, provides and a kind of prepare intelligence
The means of drug material can be transmitted.Using natural copolymer chitosan and sodium alginate as basis material, it is crosslinked using reversed-phase emulsion
Technology is acted on by intermolecular cross-linking, has obtained the temperature sensitive polymer micro-sphere material of stable structure.The present invention uses Diels-
Alder cross-linking reaction microballoon, so as to control release of the drug from microballoon by temperature, in addition preparation process avoids using
Toxic chemical crosslinking agent remains the bioactivity of natural polymer and the drug that is embedded, and significantly improves the peace of micro-sphere material
Entirely using property.Present invention process and equipment is simple, safe operation is easy, has that preparation temperature is low, curing rate is fast, process cycle
, there is application value in the advantages that short in terms of medicine controlled releasing and gene therapy.
Claims (4)
1. a kind of preparation method of temperature control drug-releasing polymer micro-sphere material, which comprises the following steps:
Chitosan is dissolved in lactic acid solution at room temperature by step 1, and succinic anhydride is added, is reacted at room temperature, and reaction solution is saturating
Water-soluble succinyl-chitosan is obtained after analysis freeze-drying；
Succinyl-chitosan is dissolved in buffer solution by step 2 at room temperature, chaff amine -2- furylamine is added dropwise into solution, so
Carbodiimides is added afterwards to be reacted, obtains furans chitosan after dialysis freeze-drying；
It is step 3, at room temperature that sodium alginate is soluble in water, sodium periodate solution is added dropwise under the conditions of being protected from light, dialyses simultaneously after reaction
Freeze-drying obtains aldehyde radical sodium alginate；
Step 4, aldehyde radical sodium alginate is soluble in water, addition 4- (4-N- maleimide phenyl) butyric acid reactant salt, dialysis
Maleimation sodium alginate is obtained after freeze-drying；
Step 5 prepares furans chitosan aqueous solution and maleimation sodium alginate aqueous solution respectively, will be matched at room temperature
Two kinds of solution of system are sufficiently mixed in isometric ratio, then the paraffin oil containing emulsifier Span 80 is added in mixed solution
In, ultrasonic emulsification obtains microballoon emulsion；
Step 6 stays overnight emulsion stirring volatilization, then pours into lotion and polymer microballoon is precipitated in isopropanol, then successively
It is cleaned respectively with isopropanol, n-hexane and acetone, finally freeze-drying obtains temperature control drug-releasing polymer micro-sphere material；
The preparation step of water solubility succinyl-chitosan described in step 1 are as follows: 0.5 gram of chitosan is dissolved in 40 milliliters of concentration
In lactic acid solution for 5% (v/v), the succinic anhydride of 1~4 times of chitosan mass is then added, it is small to stir 12~24 at room temperature
When；
The preparation of furans chitosan described in step 2 specifically: 0.5 gram of succinyl-chitosan is dissolved in 40 milliliters of phosphoric acid
In salt buffer solution, 100~500 microlitres of chaff amine -2- furylamine is then added dropwise, 1/5~1/2 times of succinyl-chitosan is added
It is stirred at room temperature after the carbodiimides of quality 12~24 hours.
2. the preparation method of temperature control drug-releasing polymer micro-sphere material according to claim 1, which is characterized in that step
The preparation of aldehyde radical sodium alginate described in 3 specifically: 1.0 grams of sodium alginate is dissolved in 100 milliliters of water, is then added dropwise
The sodium periodate solution that 2~5 milliliters of concentration are 0.5M, is stirred 1~4 hour under the conditions of being protected from light.
3. the preparation method of temperature control drug-releasing polymer micro-sphere material according to claim 1, which is characterized in that step
The preparation of maleimation sodium alginate described in 4 specifically: 1.0 grams of aldehyde radical sodium alginate is dissolved in 100 milliliters
In water, 4- (4-N- maleimide phenyl) butyric acid salting liquid that 3~12 milliliters of concentration are 0.5% is then added, at room temperature instead
It answers 0.5~2 hour.
4. the preparation method of temperature control drug-releasing polymer micro-sphere material according to claim 1, which is characterized in that step
Preparing for the 5 microballoon emulsions is specific as follows: the furans chitosan aqueous solution and maleimation sodium alginate of preparation
The concentration of aqueous solution is 0.5%~2.0% (w/v), and the volume of the emulsifier Span 80 is the 1/20~1/10 of paraffin oil,
Phaco time is 2~5 minutes.
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| "Thermogravimetric characterization of chitosan/alginate microparticles loaded with different drugs";M.I. Popa et al.;《Polymer Bulletin》;20080820;第61卷;481-490 |
| "海藻酸钠-壳聚糖固定化凝血酶微球的制备及稳定性研究";郎轶咏等;《解放军药学学报》;20120820;第28卷(第4期);311-314 |
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