CN104922694B - A kind of insulin slow release nano particle and preparation method thereof - Google Patents
A kind of insulin slow release nano particle and preparation method thereof Download PDFInfo
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- CN104922694B CN104922694B CN201510427762.4A CN201510427762A CN104922694B CN 104922694 B CN104922694 B CN 104922694B CN 201510427762 A CN201510427762 A CN 201510427762A CN 104922694 B CN104922694 B CN 104922694B
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Abstract
The present invention relates to a kind of pharmaceutical carriers, which is characterized in that it is made of five block copolymer PEI PCL PEG PCL PEI, and the drug-carrying nanometer particle prepared by the pharmaceutical carrier and active material.The active constituent can be insulin.The insulin slow release nanoparticle can discharge insulin for a long time, greatly reduce administration number of times.
Description
Technical field
The present invention relates to a kind of pharmaceutical preparations, and in particular to insulin slow release nano particle and preparation method thereof.
Background technology
Insulin is most effective Remedies for diabetes, has irreplaceable role to the treatment of diabetes, and make
It is particularly injured with that will not be generated in the process to human body.People's use of exogenous insulin diabetes have more than 80 years history,
Insulin has become key player indispensable in treating diabetes.But it since insulin is to sensitivities such as acid, heat and enzymes, takes orally
Or easily destroyed or degraded by acid, alkali and protease in gastrointestinal tract after other non-injection administrations, therefore bioavilability
It is relatively low.Therefore it is clinically conventional still using injection, the injection that diabetic need to receive long term frequent causes patient's compliance low.
In recent years, it takes orally or the exploitation of other insulin preparations easy to use has caused the concern of domestic and international researcher, but this kind of system
Agent need to further solve medicine-feeding part or drug release position mucous membrane transmitance it is low, drug absorption is poor, drug hydrolysis, liver caused by body fluid
The problems such as dirty first pass effect.Therefore, it to obtain hypoglycemic effect and the higher bioavilability with therapeutic potential, injects
Administration still belongs to preferred administration route.
In order to effectively play insulin function and reach good therapeutic effect, it is necessary to select suitable pharmaceutical carrier, medicine
Agent type and administration route, enable body effectively to absorb the drug.In terms of injection of insulin agent, main slow-release controlled-release material
Material has injectable nanoparticle and injectable gel etc..Dong bank outstanding person etc.[1]A kind of temperature by chemical bond carrying medicament of disclosure of the invention
Quick situ-gel and preparation.The gel is to be scattered in shape in aqueous systems by the polyethylene glycol ester block copolymer of bonding drug
It is and the polyester block B by ethylene glycol block A at, the block copolymer, and C/B-A-B/C types, A-B/C is constituted with drug C
The copolymer of type, C/A-B types or C/A-B/C types.Preparation process is simple, and the temperature sensing in situ gel rubber of carrying medicament has a variety of prodrugs
Application prospect.Soup morning sunlight etc.[2]Provide a kind of insulin drug carried microspheres insulin drug carried microspheres provided by the invention take orally to
When medicine, it can realize release and absorption of the insulin in enteron aisle, the bioavilability of insulin can be improved.Zhu Limin etc.[3]
A kind of spontaneously-combined chitosan medicine-carrying nano particle and preparation method thereof is invented, the nanoparticle is by core sheath electrospinning composite fibrofelt in water
In spontaneous assembling formed, it is chitosan that nanoparticle, which has nucleocapsid, the shell of nanoparticle, and kernel is drug, is had certain
Slowly releasing effect.
Herein respectively with five block polymer A, that is, PEI10000-PCL4000-PEG2000-PCL4000-PEI10000, five blocks it is poly-
Close object B, that is, PEI10000-PCL5000-PEG2000-PCL5000-PEI10000With five block polymer C, that is, PEI10000-PCL6000-
PEG2000-PCL6000-PEI10000For carrier material, the load insulin nanoparticles with slowly releasing effect are prepared for, as pancreas islet
Essence injecta use aspect has certain application prospect.
Invention content
One aspect of the invention is related to a kind of pharmaceutical carrier, by five block copolymer PEI-PCL-PEG-PCL-PEI systems
At, it is preferable that the five block copolymers PEI-PCL-PEG-PCL-PEI is PEIa-PCLb-PEGc-PCLd-PEIe, therein
A, b, c, d, e are respectively 2000-20000, and more electedly, a and e are respectively 10000, and the c is 2000, and the b and d divide
Not Wei 4000-6000, most preferably, the five block copolymers PEI-PCL-PEG-PCL-PEI be PEI10000-PCL4000-
PEG2000-PCL4000-PEI10000, PEI10000-PCL5000-PEG2000-PCL5000-PEI10000Or PEI10000-PCL6000-PEG2000-
PCL6000-PEI10000。
Further, the pharmaceutical carrier be by by the five block copolymers PEI-PCL-PEG-PCL-PEI to have
Machine solution dissolves, and is then added drop-wise in aqueous solution, stirs and form nano-solution.
It includes macromolecular polypeptides or micromolecular compound that another aspect of the invention, which is related to foregoing pharmaceutical carrier preparing,
Application in drug.
Another aspect of the invention is related to the preparation method of foregoing pharmaceutical carrier, and it includes following steps：
1) synthesis triblock polymer PCLb-PEGc-PCLd
PCL is synthesized using ring-opening polymerisation methodb-PEGc-PCLdCopolymer, with PEGcIt is reacted with ε-CL, and with 1%mol
ε-CL amount using stannous octoate as catalyst；
2) PCL of acrylic ester synthesizing sealing endb-PEGc-PCLd
By PCL obtained by step 1)b-PEGc-PCLdIt is dissolved with organic solvent, then is separately added into triethylamine and acryloyl chloride (three
Ethamine:Acryloyl chloride:PCLb-PEGc-PCLd=4:4.2:1), stirring is filtered to room temperature after reaction, is down at 80 DEG C
Remove triethylamine hydrochloride crystallization；Filtered liquid precipitates in the n-hexane of excessive ice, and acrylate envelope is collected after suction filtration
The PCL at endb-PEGc-PCLd；
3) five block polymer PEI are synthesizeda-PCLb-PEGc-PCLd-PEIe
PEI is synthesized using Michael addition reactionsa-PCLb-PEGc-PCLd-PEIe, respectively by PEI and acrylate ended
PCLb-PEGc-PCLdBe dissolved in organic solvent respectively, by PEI chloroformic solutions under the conditions of 60 DEG C stirring and dissolving, then to PEI chlorine
The PCL of acrylate ended is added dropwise in imitative solutionb-PEGc-PCLdChloroformic solution vacuumizes logical nitrogen and holds afterwards in triplicate
It is continuous to be stirred at reflux reaction for 24 hours, after reaction, it is down to room temperature, crude product precipitates in excessive cold petroleum ether, and white is obtained by filtration
Wax depositions, product is dissolved with q. s. methylene chloride, and acquired solution is instilled to stirring in deionized water dropwise and forms milky
Solution is stirred overnight five block polymer PEI of freeze-drying acquisition after volatile organic solventa-PCLb-PEGc-PCLd-PEIe。
Another aspect of the invention is related to a kind of insulin slow release nano particle, which is characterized in that it is by five block copolymerizations
The pharmaceutical carrier of object PEI-PCL-PEG-PCL-PEI contains insulin and is made.
Further, the five block copolymers PEI-PCL-PEG-PCL-PEI is PEIa-PCLb-PEGc-PCLd-PEIe,
A, b, c, d, e therein are respectively 2000-20000.
Further, a and e is respectively 8000-12000, and the c is 1500-2500, and the b and d are respectively
3000-7000。
Further, a and e is respectively 10000, and the c is 2000, and the b and d are respectively 4000-6000.
Further, the five block copolymers PEI-PCL-PEG-PCL-PEI is PEI10000-PCL4000-PEG2000-
PCL4000-PEI10000, PEI10000-PCL5000-PEG2000-PCL5000-PEI10000Or PEI10000-PCL6000-PEG2000-PCL6000-
PEI10000。
Further, the mass ratio (w/w) of the insulin and pharmaceutical carrier be 10-50%, preferably 20-40%, more
Preferably 20%, 25%, 30%, 35%, 40%.
Another aspect of the invention is related to aforementioned slow release nano-particle and is preparing the application in treating metabolic disease, preferably
Ground, the metabolic disease are selected from diabetes, hyperglycemia, more preferably type-1 diabetes mellitus or type-2 diabetes mellitus.
Another aspect of the invention is related to the preparation method of aforementioned slow release nano-particle, includes the following steps：
(1) active ingredient solution is prepared,
(2) pharmaceutical carrier is dissolved in organic solvent,
(3) active ingredient solution is added dropwise in pharmaceutical carrier solution, stirring, volatile organic solvent,
(4) it centrifuges and collects precipitation to get polymer drug-carried slow release nano-particle.
Further, the step is：
(1) precision weighs 4.0mg insulin, is dissolved in 0.01mol/L HCl solutions, then molten with a small amount of 1mol/L NaOH
Liquid adjusts its pH value to 5.35 or more；
(2) pharmaceutical carrier is dissolved in dichloromethane organic solvent；
(3) (10s/d) is added dropwise under 400r/min magnetic agitations dropwise to above-mentioned insulin solutions, after 1~2h is stirred at room temperature
4h is persistently stirred under the conditions of 40 DEG C again, organic solvent is made to volatilize naturally；
(4) 1h is centrifuged under the conditions of 14000rpm/min and collects precipitation, and is freeze-dried after being washed with deionized water three times,
Up to polymer drug-carried slow release nano-particle.
Further, the pH value wherein in step (1) is adjusted to 5.8-6.2, preferably 6.
Description of the drawings
Fig. 1 is the synthesis step of five block polymer PEI-PCL-PEG-PCL-PEI：
Figure 1A is the synthesis schematic diagram of triblock polymer PCL-PEG-PCL.
Figure 1B is the synthesis schematic diagram of the PCL-PEG-PCL of acrylate ended.
Fig. 1 C are the synthesis schematic diagram of five block polymer PEI-PCL-PEG-PCL-PEI.
Fig. 2 is the FT-IR spectrograms that product is respectively walked in three step synthetic polymer A：
(a) triblock polymer PCL4000-PEG2000-PCL4000FT-IR spectrograms.
(b) PCL of acrylate ended4000-PEG2000-PCL4000FT-IR spectrograms.
(c) five block polymer PEI10000-PCL4000-PEG2000-PCL4000-PEI10000FT-IR spectrograms.
Fig. 3 is the FT-IR spectrograms that product is respectively walked in three step synthetic polymer B：
(a) triblock polymer PCL5000-PEG2000-PCL5000FT-IR spectrograms.
(b) PCL of acrylate ended5000-PEG2000-PCL5000FT-IR spectrograms.
(c) five block polymer PEI10000-PCL5000-PEG2000-PCL5000-PEI10000FT-IR spectrograms.
Fig. 4 is the FT-IR spectrograms that product is respectively walked in three step synthetic polymer C：
(a) triblock polymer PCL6000-PEG2000-PCL6000FT-IR spectrograms.
(b) PCL of acrylate ended6000-PEG2000-PCL6000FT-IR spectrograms.
(c) five block polymer PEI10000-PCL6000-PEG2000-PCL6000-PEI10000FT-IR spectrograms.
Fig. 5 is respectively to walk product in three step synthetic polymer A1HNMR spectrograms：
Fig. 5 A are triblock polymer PCL4000-PEG2000-PCL4000's1H-NMR spectrum.
Fig. 5 B are the PCL of acrylate ended4000-PEG2000-PCL4000's1HNMR spectrograms.
Fig. 5 C are five block polymer PEI10000-PCL4000-PEG2000-PCL4000-PEI10000's1HNMR spectrograms.
Fig. 6 is respectively to walk product in three step synthetic polymer B1HNMR spectrograms：
Fig. 6 A are triblock polymer PCL5000-PEG2000-PCL5000's1H-NMR spectrum.
Fig. 6 B are the PCL of acrylate ended5000-PEG2000-PCL5000's1HNMR spectrograms.
Fig. 6 C are five block polymer PEI10000-PCL5000-PEG2000-PCL5000-PEI10000's1HNMR spectrograms.
Fig. 7 is respectively to walk product in three step synthetic polymer C1HNMR spectrograms：
Fig. 7 A are triblock polymer PCL6000-PEG2000-PCL6000's1H-NMR spectrum.
Fig. 7 B are the PCL of acrylate ended6000-PEG2000-PCL6000's1HNMR spectrograms.
Fig. 7 C are five block polymer PEI10000-PCL6000-PEG2000-PCL6000-PEI10000's1HNMR spectrograms.
Fig. 8 is fluorescence excitation spectrum (hair of the pyrene in various concentration PEI-PCL-PEG-PCL-PEI nanoparticle aqueous dispersions
Penetrate a length of 393nm of light wave).
Fig. 8 A are pyrene in various concentration PEI10000-PCL4000-PEG2000-PCL4000-PEI10000In nanoparticle aqueous dispersions
Fluorescence excitation spectrum (wavelength of transmitted light 393nm).
Fig. 8 B are pyrene in various concentration PEI10000-PCL5000-PEG2000-PCL5000-PEI10000In nanoparticle aqueous dispersions
Fluorescence excitation spectrum (wavelength of transmitted light 393nm).
Fig. 8 C are pyrene in various concentration PEI10000-PCL6000-PEG2000-PCL6000-PEI10000In nanoparticle aqueous dispersions
Fluorescence excitation spectrum (wavelength of transmitted light 393nm).
Fig. 9 is the fluorescence excitation luminous intensity ratio I of pyrene337/I334It is dense with PEI-PCL-PEG-PCL-PEI nanoparticle aqueous dispersions
Spend logarithmic relationship figure.
Fig. 9 A are the fluorescence excitation luminous intensity ratio I of pyrene337/I334With PEI10000-PCL4000-PEG2000-PCL4000-PEI10000
Nanoparticle aqueous dispersions log concentration relational graph.
Fig. 9 B are the fluorescence excitation luminous intensity ratio I of pyrene337/I334With PEI10000-PCL5000-PEG2000-PCL5000-PEI10000
Nanoparticle aqueous dispersions log concentration relational graph.
Fig. 9 C are the fluorescence excitation luminous intensity ratio I of pyrene337/I334With PEI10000-PCL6000-PEG2000-PCL6000-PEI10000
Nanoparticle aqueous dispersions log concentration relational graph.
Figure 10 is the transmission electron microscope picture of polymer supported insulin nanoparticles.
Figure 10 A are INS-PEI10000-PCL4000-PEG2000-PCL4000-PEI10000The transmission electron microscope picture of nanoparticle.
Figure 10 B are INS-PEI10000-PCL5000-PEG2000-PCL5000-PEI10000The transmission electron microscope picture of nanoparticle.
Figure 10 C are INS-PEI10000-PCL5000-PEG2000-PCL5000-PEI10000The transmission electron microscope picture of nanoparticle.
Figure 11 is the In-vitro release curves of polymer supported insulin nanoparticles
Figure 12 is that the change of blood sugar figure after polymer supported insulin nanoparticles is subcutaneously injected in diabetic mice.(n=3,
x±s)。
Specific implementation mode
The synthesis of 1 insulin carrier material of embodiment, five block polymer PEI-PCL-PEG-PCL-PEI
Embodiment 1-1 synthesis triblock polymers PCL-PEG-PCL
PCL-PEG-PCL triblock copolymers, the appropriate PEG of precision weighing are synthesized using ring-opening polymerisation method2000With ε-CL, set
In the drying round-bottomed flask of 50ml, using the stannous octoate of the ε-CL amounts of 1%mol as catalyst.Reaction unit vacuumizes logical
Nitrogen, oil bath heating is stirred to react for 24 hours under the conditions of 125 DEG C after the repetition operation three times.Wait for that reaction solution is slightly cold after reaction,
Reaction solution is dissolved with q. s. methylene chloride, is added dropwise under agitation in excessive ice ether and obtains white precipitate product,
It filters and collects white powder product.It repeats aforesaid operations and product is dried under vacuum to constant weight under the conditions of 40 DEG C afterwards three times.
Reaction equation is as shown in Figure 1A.
The PCL-PEG-PCL of embodiment 1-2 acrylic ester synthesizings sealing end
0.1mmol PCL-PEG-PCL are placed in 50ml round-bottomed flasks, 16ml toluene stirring and dissolvings are used in combination.Add respectively again
Enter 0.40mmol triethylamines and 0.42mmmol acryloyl chloride (triethylamines:Acryloyl chloride:Triblock polymer=4:4.2:1), instead
It answers device to vacuumize logical nitrogen, 8h is stirred to react at 80 DEG C after repeating the operation three times.After reaction, it is down to room temperature, instead
Liquid is answered to be filtered to remove triethylamine hydrochloride crystallization.Filtered liquid precipitates in the n-hexane of excessive ice, is collected after suction filtration white
Color powdery product.It repeats aforesaid operations and product is dried under vacuum to constant weight under the conditions of 40 DEG C afterwards twice.Reaction equation such as Figure 1B
It is shown.
Embodiment 1-3 synthesizes five block polymer PEI-PCL-PEG-PCL-PEI
PEI-PCL-PEG-PCL-PEI is synthesized using Michael addition reactions.Respectively by 1mmolPEI10000With 1mmol third
The PCL-PEG-PCL of olefin(e) acid ester sealing end is dissolved in appropriate chloroform respectively.First by PEI chloroformic solutions in 60 DEG C, atmosphere of inert gases
Stirring is to being completely dissolved, then the PCL-PEG-PCL chloroformic solutions of acrylate ended are added dropwise to above-mentioned solution, vacuumizes logical
After nitrogen repeats the operation three times, reaction is persistently stirred at reflux for 24 hours under the conditions of 60 DEG C.After reaction, it is down to room temperature, it is thick to produce
Object precipitates in excessive cold petroleum ether, and White waxy precipitation is obtained by filtration.After product is dissolved with q. s. methylene chloride, gained is molten
Liquid instills stirring in deionized water and forms milky white solution dropwise, is freeze-dried after being stirred overnight volatile organic solvent.Reaction equation
As shown in Figure 1 C.
The characterization of 2 synthetic product of embodiment
Embodiment 2-1 infrared spectrum characterizations are analyzed
Embodiment 2-1-1 PEI10000-PCL4000-PEG2000-PCL4000-PEI10000Infrared spectrum characterization is analyzed
By the PCL-PEG-PCL polymerizations of PCL-PEG-PCL triblock polymers, acrylate ended on a small quantity after purification
After object, five block polymers of PEI-PCL-PEG-PCL-PEI are milled into powder using potassium bromide as dispersant at room temperature, take appropriate
Sample tabletting is in 400~4000cm-1Wave number scans, and measures its infrared absorption spectrum.As shown in Fig. 2, 1109cm in curve a-1Place
For the stretching vibration peak of the C-O keys of PEG, 2867cm-1Place is C-H stretching vibration peaks；1725cm-1Place is PCL segment C=O keys
Stretching vibration peak, 3438cm-1Peak be PCL segments end-OH stretching vibration peak.Compared with PCL-PEG-PCL, curve b
There is stronger 1636cm in the infrared spectrum of the PCL-PEG-PCL of middle acrylate ended-1The absorption at place, 1636cm-1Ownership
In the stretching vibration absworption peak of-C=C-, illustrate that intermediate synthesizes successfully.Occurs stronger 3404cm in curve c-1The suction at place
It receives, belongs to the stretching vibration characteristic absorption peak of amino on PEI, infrared spectrum result is consistent with the expected structure of polymer.
Embodiment 2-1-2 PEI10000-PCL5000-PEG2000-PCL5000-PEI10000Infrared spectrum characterization is analyzed
As shown in figure 3,1107cm in curve a-1Place is the stretching vibration peak of the C-O keys of PEG, 2866cm-1It is stretched for C-H at place
Contracting vibration peak；1725cm-1Place is the stretching vibration peak of PCL segment C=O keys, 3437cm-1Peak be PCL segments end-OH
Stretching vibration peak.Compared with PCL-PEG-PCL, the infrared spectrum of the PCL-PEG-PCL of acrylate ended occurs in curve b
Stronger 1636cm-1The absorption at place, 1636cm-1The stretching vibration absworption peak for belonging to-C=C-, illustrate intermediate synthesis at
Work(.Occurs stronger 3392cm in curve c-1The absorption at place belongs to the stretching vibration characteristic absorption peak of amino on PEI, red
Outer spectrogram result is consistent with the expected structure of polymer.
Embodiment 2-1-3 PEI10000-PCL6000-PEG2000-PCL6000-PEI10000Infrared spectrum characterization is analyzed
As shown in figure 4,1108cm in curve a-1Place is the stretching vibration peak of the C-O keys of PEG, 2866cm-1It is stretched for C-H at place
Contracting vibration peak；1724cm-1Place is the stretching vibration peak of PCL segment C=O keys, 3438cm-1Peak be PCL segments end-OH
Stretching vibration peak.Compared with PCL-PEG-PCL, the infrared spectrum of the PCL-PEG-PCL of acrylate ended occurs in curve b
Stronger 1636cm-1The absorption at place, 1636cm-1The stretching vibration absworption peak for belonging to-C=C-, illustrate intermediate synthesis at
Work(.Occurs stronger 3389cm in curve c-1The absorption at place belongs to the stretching vibration characteristic absorption peak of amino on PEI, red
Outer spectrogram result is consistent with the expected structure of polymer.
Embodiment 2-21H-NMR phenetic analysis
Embodiment 2-2-1 PEI10000-PCL4000-PEG2000-PCL4000-PEI10000 1H-NMR phenetic analysis
By the PCL-PEG-PCL polymerizations of PCL-PEG-PCL triblock polymers, acrylate ended on a small quantity after purification
Object, five block polymers of PEI-PCL-PEG-PCL-PEI point are dissolved in deuterochloroform (CDCl3) in, made with tetramethylsilane (TMS)
For internal standard compound, carry out1H-NMR spectrum (400MHz) characterize.It is illustrated in fig. 5 shown below, δ=3.66ppm is in macromonomer in b figures
The characteristic peak of the methylene hydrogen of PEG units, δ=2.33ppm are that caprolactone units are connected-CH with carbonyl in macromonomer2-
Characteristic peak, the characteristic peak of other methylene hydrogen of caprolactone units in δ=1.40ppm and 1.67ppm macromonomers.δ=
5.85ppm and 6.40ppm is the characteristic peak of two β-H of acrylate ended part respectively, and 6.14ppm is acrylate ended
The characteristic peak of partial α-H illustrates the PCL for successfully having synthesized intermediate-acrylate ended of reaction4000-PEG2000-
PCL4000.- CH=CH in c figures2The characteristic peak of two β-H and the characteristic peak of α-H do not occur, illustrate react third step product
The middle PCL without unreacted acrylate ended4000-PEG2000-PCL4000Macromonomer.δ=4.08ppm be PCL units (-
OCH2-CH2-CH2-CH2-CH2CO--the CH being connected with oxygen in)2Characteristic peak, C δ=2.33ppm be connected with carbonyl-
CH2Characteristic peak.δ=3.67ppm is the characteristic peak of the methylene hydrogen of peg moiety in macromonomer.Chemical shift is 2.50
~2.86ppm is PEI units (- NHCH2CH2) methylene hydrogen characteristic peak, illustrate successfully to have synthesized PEI10000-
PCL4000-PEG2000-PCL4000-PEI10000Five block polymers.The ratio of polymer P EG/PEI second from PEG in Fig. 5-c
Peak area (4H, the δ 3.67ppm ,-CH of the hydrogen atom of support group group2CH2O-) and in PEI the hydrogen atom of ethylene group peak area (4H,
2.50~2.86ppm ,-CH2CH2ONH- ratio) is calculated.The ratio of polymer P EG/PCL ethylene from PEG in Fig. 5-c
Peak area (4H, the δ 3.67ppm ,-CH of the hydrogen atom of group2CH2O- the methylene hydrogen atom) and in PCL blocks being connected with oxygen
The ratio of peak area (2H, δ 4.08ppm ,-OCH2-) is calculated.Due to PEG molecular weight it is known that be 2000, polymerization
Object PEI10000-PCL4000-PEG2000-PCL4000-PEI10000Molecular weight can be calculated by the ratio of PEG/PEI and PEG/PCL
It obtains.Gained PEI is calculated according to above-mentioned formula10000-PCL4000-PEG2000-PCL4000-PEI10000Five block polymers it is opposite
Molecular mass is respectively 302550, close with calculated value.
Embodiment 2-2-2 PEI10000-PCL5000-PEG2000-PCL5000-PEI10000 1H-NMR phenetic analysis
It is illustrated in fig. 6 shown below, δ=3.66ppm is the characteristic peak of the methylene hydrogen of PEG units in macromonomer, δ in b figures
=2.30ppm is that caprolactone units are connected-CH with carbonyl in macromonomer2Characteristic peak, δ=1.40ppm and
The characteristic peak of other methylene hydrogen of caprolactone units in 1.67ppm macromonomers.δ=5.86ppm and 6.39ppm is third respectively
The characteristic peak of two β-H of olefin(e) acid ester end section, 6.14ppm are the characteristic peaks of the α-H of acrylate ended part, are illustrated as
Work(has synthesized the PCL of intermediate-acrylate ended of reaction5000-PEG2000-PCL5000.- CH=CH in c figures2Two
The characteristic peak of β-H and the characteristic peak of α-H do not occur, illustrate to react in the product that third walks without unreacted acrylate ended
PCL5000-PEG2000-PCL5000Macromonomer.δ=4.06ppm is PCL units (- OCH2-CH2-CH2-CH2-CH2CO- in) with
Connected-the CH of oxygen2Characteristic peak, C δ=2.19ppm are the-CH that is connected with carbonyl2Characteristic peak.δ=3.66ppm is big
The characteristic peak of the methylene hydrogen of peg moiety in molecule monomer.Chemical shift be 2.22~2.47ppm be PEI units (-
NHCH2CH2) methylene hydrogen characteristic peak, illustrate successfully to have synthesized PEI10000-PCL5000-PEG2000-PCL5000-
PEI10000Five block polymers.The peak face of the ratio of polymer P EG/PEI hydrogen atom of ethylene group from PEG in Fig. 6-c
Product (4H, δ 3.66ppm ,-CH2CH2O-) and in PEI the hydrogen atom of ethylene group peak area (4H, 2.40~2.90ppm, --
CH2CH2ONH- ratio) is calculated.The ratio of polymer P EG/PCL hydrogen atom of ethylene group from PEG in Fig. 6-c
Peak area (4H, δ 3.66ppm ,-CH2CH2O- peak area (2H, the δ for the methylene hydrogen atom) and in PCL blocks being connected with oxygen
4.06ppm ,-OCH2-) ratio be calculated.Due to PEG molecular weight it is known that be 2000, polymer P EI10000-
PCL5000-PEG2000-PCL5000-PEI10000Molecular weight can be calculated by the ratio of PEG/PEI and PEG/PCL.According to upper
It states formula and calculates gained PEI10000-PCL5000-PEG2000-PCL5000-PEI10000The relative molecular mass of five block polymers point
Not Wei 30299, it is close with calculated value.
Embodiment 2-2-3 PEI10000-PCL6000-PEG2000-PCL6000-PEI10000 1H-NMR phenetic analysis
It is illustrated in fig. 7 shown below, δ=3.66ppm is the characteristic peak of the methylene hydrogen of PEG units in macromonomer, δ in b figures
=2.31ppm is that caprolactone units are connected-CH with carbonyl in macromonomer2Characteristic peak, δ=1.40ppm and
The characteristic peak of other methylene hydrogen of caprolactone units in 1.67ppm macromonomers.δ=5.85ppm and 6.40ppm is third respectively
The characteristic peak of two β-H of olefin(e) acid ester end section, 6.17ppm are the characteristic peaks of the α-H of acrylate ended part, are illustrated as
Work(has synthesized the PCL of intermediate-acrylate ended of reaction6000-PEG2000-PCL6000.- CH=CH in c figures2Two
The characteristic peak of β-H and the characteristic peak of α-H do not occur, illustrate to react in the product that third walks without unreacted acrylate ended
PCL6000-PEG2000-PCL6000Macromonomer.δ=4.06ppm is PCL units (- OCH2-CH2-CH2-CH2-CH2CO- in) with
Connected-the CH of oxygen2Characteristic peak, C δ=2.31ppm are the-CH that is connected with carbonyl2Characteristic peak.δ=3.67ppm is big
The characteristic peak of the methylene hydrogen of peg moiety in molecule monomer.Chemical shift be 2.42~2.97ppm be PEI units (-
NHCH2CH2) methylene hydrogen characteristic peak, illustrate successfully to have synthesized PEI10000-PCL6000-PEG2000-PCL6000-
PEI10000Five block polymers.The peak face of the ratio of polymer P EG/PEI hydrogen atom of ethylene group from PEG in Fig. 6-c
Product (4H, δ 3.67ppm ,-CH2CH2O-) and in PEI the hydrogen atom of ethylene group peak area (4H, 2.42~2.97ppm, --
CH2CH2ONH- ratio) is calculated.The ratio of polymer P EG/PCL hydrogen atom of ethylene group from PEG in Fig. 7-c
Peak area (4H, δ 3.67ppm ,-CH2CH2O- peak area (2H, the δ for the methylene hydrogen atom) and in PCL blocks being connected with oxygen
4.07ppm ,-OCH2-) ratio be calculated.Due to PEG molecular weight it is known that be 2000, polymer P EI10000-
PCL6000-PEG2000-PCL6000-PEI10000Molecular weight can be calculated by the ratio of PEG/PEI and PEG/PCL.According to upper
It states formula and calculates gained PEI10000-PCL6000-PEG2000-PCL6000-PEI10000The relative molecular mass of five block polymers point
Not Wei 33735, it is close with calculated value.
The preparation and representation of 3 blank polymer nanoparticle of embodiment
Polymer A:PEI10000-PCL4000-PEG2000-PCL4000-PEI10000
Polymer B:PEI10000-PCL5000-PEG2000-PCL5000-PEI10000
Polymer C:PEI10000-PCL6000-PEG2000-PCL6000-PEI10000
Blank polymer nanoparticle is prepared using solvent evaporation method, the polymer A/B/C of 10/12/20mg is dissolved in 500ul
Dichloromethane organic solvent is added dropwise in (10s/d) to 10mL deionized waters under 400r/min magnetic agitations, is stirred at room temperature 1 dropwise
4h is persistently stirred after~2h under the conditions of 40 DEG C, organic solvent obtains blank polymer nanoparticle after volatilizing naturally, and carries out phase
The detection answered.Grain size, the dispersity index (PDI) of particle are investigated with Malvern laser particle analyzer.It the results are shown in Table 1.
1 PEI-PCL-PEG-PCL-PEI polymer of table and its characterization of formation
The preparation of 4 polymer drug-carried nanoparticle of embodiment
Polymer A:PEI10000-PCL4000-PEG2000-PCL4000-PEI10000
Polymer B:PEI10000-PCL5000-PEG2000-PCL5000-PEI10000
Polymer C:PEI10000-PCL6000-PEG2000-PCL6000-PEI10000
Precision weighs 4.0mg insulin, is dissolved in the 0.01mol/L HCl solutions of 10mL, then with a small amount of 1mol/LNaOH
Solution adjust its pH value to 6.0 (insulin solutions when insulin isoelectric pH=5.30~5.35, pH are more than insulin isoelectric point
It is negatively charged, electrostatical binding can occur with the polymer material of grafting PEI).The polymer A/B/C of 10/12/20mg is dissolved in
(10/d) is added dropwise under 400r/min magnetic agitations to above-mentioned 10mL insulin solutions, room dropwise for 500ul dichloromethane organic solvents
4h is persistently stirred under the conditions of 40 DEG C again after 1~2h of temperature stirring, in 14000rpm/min conditions after making organic solvent volatilize naturally
Lower centrifugation 1h simultaneously collects precipitation, and is freeze-dried to get polymer drug-carried nanoparticle after being washed with deionized water three times.
The measurement of 5 critical aggregation concentration CAC values of embodiment
The CAC values of blank polymer nanoparticle are measured using fluorescence probe method.With 1mgmL-1Polymer water disperse system
Configuration 0.5-5 × 10 are diluted for mother liquor-5mg·mL-1Appropriate pyrene-acetone soln is added in totally 9 various concentration solution so that every
A concentration of the 5.93 × 10 of pyrene in pipe-7mol·L-1, 50。After C forced air dryings eliminate acetone, 65。C water bath with thermostatic control 3h keep pyrene solubilized
It is stood overnight into polymer nanoparticle postcooling, it is to be measured.It is scanned under the conditions of launch wavelength 393nm, EX and EM width 3nm
300-370nm ranges record the fluorescence spectra of pyrene under various concentration, and with log (C, mgmL-1) with pyrene in 334nm and
Intensity rate (I at 337nm337/I334) mapping, low concentration and the be in line inflection point of high concentration are the polymer nanoparticle
Critical aggregation concentration CAC values.The CAC values of polymer A/B/C are respectively 10.96 × 10-4g·L-1、8.13×10-4g·L-1、
6.17×10-4g·L-1, the hydrophobic segment of polymer is bigger, and CAC values are smaller, and the structure being self-assembly of is more stable.Pyrene exists
The fluorescence excitation luminous intensity ratio I of fluorescence excitation spectrum and pyrene in various concentration polymer A/B/C solution337/I334With polymer
A/B/C concentration of aqueous solution logarithmic relationship figures are shown in Fig. 8 and Fig. 9.
The measurement of 6 INS-PEI-PCL-PEG-PCL-PEI nanoparticles encapsulation rate of embodiment and drugloading rate
The drafting of embodiment 6-1 standard curves
Precision weighs 25.0mg insulin, is dissolved in 0.01mol/L HCl solutions, is settled to 25mL, is made into a concentration of
The insulin mother liquor of 1.00mg/mL.It is accurate respectively to draw mother liquor 0.1,0.2,0.4,0.5,0.6,0.8,1.0,1.5,2.0mL extremely
In 10mL volumetric flasks, 0.01mol/L HCl solutions are settled to 10mL, obtain the solution to be measured of 9 mass concentrations.Correct amount respectively
Above-mentioned sample 1.0mL is taken, Coomassie brilliant G-250 reagent 4.0mL is added thereto, shakes up, using 0.01mol/L HCl solutions
It is blank control with sample made from method, in 10min to 1h, is measured in maximum absorption wave strong point on ultraviolet specrophotometer
Absorbance value A.The absorbance value that experiment is measured and corresponding insulin concentration relationship linear fit, obtain standard curve y=
0.0049x+0.1266,R2=0.9951.
The measurement of embodiment 6-2 drug-carrying nanometer particles encapsulation rate and drugloading rate
Respectively by the different 10ml blank nanoparticles for carrying medicine ratio and drug-carrying nanometer particle solution in 25000rpm/min conditions
Supernatant is collected after lower centrifugation 1h.1.0ml supernatants are taken, the colour developing of 4.0ml Coomassie Brillant Blue solutions is added, is stood after mixing
10min is control with the supernatant of blank nanoparticle solution, with the nanoparticle of the incomplete grain size very little centrifuged of elimination
It influences.The absorbance for measuring drug-carrying nanometer particle solution supernatant on ultraviolet specrophotometer at 595nm, according to insulin standard
The concentration of insulin is calculated in curve, calculates drug-carrying nanometer particle encapsulation rate and carrying drug ratio further according to following formula, the results are shown in Table 2.
Encapsulation rate (EE%)=(quality of gross mass-free insulin of insulin)/dispensing gross mass × 100%
The gross mass of carrying drug ratio (DL%)=(quality of gross mass-free insulin of insulin)/nanometer formulation ×
100%
With SPSS20.0 statistical analysis softwares between the different dispensings of polymer A/B/C drug-carrying nanometer particles than the encapsulation rate group
Make One-way ANOVA, P respectively with drugloading rate<0.05 is to have notable significant difference.Polymer A drug-carrying nanometer particle differences are offerd medicine
Than encapsulation rate between group, there are statistical discrepancy (P=0.027)；The dispensing of polymer B drug-carrying nanometer particle difference exists than encapsulation rate between group
Statistical discrepancy (P=0.001)；There are statistical discrepancy (P=than encapsulation rate between group for the dispensing of polymer C drug-carrying nanometer particle differences
0.001).There are statistical discrepancy (P=0.000) than drugloading rate between group for the dispensing of polymer A drug-carrying nanometer particle differences；Polymer B carries
There are statistical discrepancy (P=0.000) than drugloading rate between group for the dispensing of medicine nanoparticle difference；Polymer C drug-carrying nanometer particle differences are offerd medicine
Than drugloading rate between group, there are statistical discrepancy (P=0.000)；The drugloading rate of polymer drug-carried nanoparticle can more reflect carrier to drug
Embedding situation, therefore for drugloading rate, polymer A drug-carrying nanometer particles 20%wt dispensings are than group with 33%wt dispensings than organizing it
Between there are statistical discrepancy (P=0.000), 20%wt dispensings are than there are statistical discrepancy (P=between group and 40%wt dispensing ratios
0.000), 33%wt dispensings are offerd medicine than group and 40%wt than there are statistical discrepancy (P=0.001) between group, therefore follow-up test is selected
Select this dispensing ratio of 40%wt.Similarly, polymer B drug-carrying nanometer particle and polymer C drug-carrying nanometer particles select 40%wt this
Dispensing ratio carries out load medicine.
2 polymer drug-carried nanoparticle encapsulation rate of table and drugloading rate
The transmission electron microscope picture of 7 polymer drug-carried nanoparticle of embodiment
The polymer drug-carried nanoparticle form prepared using transmission electron microscope observing.Proper amount of nano grain solution is taken, is diluted to one
It after determining concentration, drops to and is covered on the copper mesh of carbon film, stop 2min, suck redundant solution with filter paper, it is negative that 3% Salkowski's solution is added dropwise
2min is contaminated, is dried under infrared lamp, transmission electron microscope observing is taken pictures.The hydrophobic of polymer drug-carried nanoparticle is formed in transmission electron microscope picture
Segment part is not dyed by phosphotungstic acid, is in brilliant white kernel, and hydrophilic polyethylene glycol shell has adsorbed the part phosphorus tungsten in water phase
Acid and under Electronic Speculum be in grey black halo.Polymer A/B/C drug-carrying nanometer particle transmission electron microscope pictures clearly show that spherical junctions
Structure；In addition, drug-carrying nanometer particle grain size shown in transmission electron microscope picture is respectively less than the nanoparticle grain size that Malvern particle instrument measures,
Possible cause is that the grain size that Malvern particle instrument measures is the result that multiple drug-carrying nanometer particles are sticked together.It is polymer drug-carried to receive
Grain of rice transmission electron microscope picture is shown in Figure 10 A- Figure 10 C.
The tablets in vitro of 8 polymer drug-carried nanoparticle of embodiment is investigated
The polymer A/B/C drug-carrying nanometer particle 5mg after freeze-drying are taken, totally nine parts, are respectively placed in the PBS buffer solution of 5ml
(0.01mol·l-1, pH=6.0) in, it is transferred in 50ml centrifuge tubes, 37 DEG C of waters bath with thermostatic control, in 70rmin-1At the uniform velocity stir,
Keep sink conditions.1mL solution, 14000rmin are taken out when 0,0.5,1,2,4,6,8,12,24,36,48h-1At a high speed
30min is centrifuged, takes supernatant to measure insulin content, and continue to release after supplementing the fresh medium to 50mL centrifuge tubes of same volume
Medicine, the Cumulative release amount in the computational rules time.Using drug release time as abscissa, it is ordinate to add up release rate, draws polymerization
The In-vitro release curves of object A/B/C drug-carrying nanometer particles.From release profiles it can be seen that polymer A/B/C drug-carrying nanometer particles have obviously
Burst effect, preceding 8h is in the quick release phase, for polymer A drug-carrying nanometer particles, accumulative release of the insulin in this stage
Amount reaches 34.92%；For polymer B drug-carrying nanometer particle, Cumulative release amount of the insulin in this stage reaches 25.03%；It is right
In polymer C drug-carrying nanometer particles, Cumulative release amount of the insulin in this stage reaches 16.46%；For polymer A medicament-carried nanos
Grain, the Cumulative release amount at the ends insulin 48h reach 49.92%；For polymer B drug-carrying nanometer particle, the ends insulin 48h add up
Burst size reaches 33.17%；For polymer C drug-carrying nanometer particles, the Cumulative release amount at the ends insulin 48h reaches 24.36%.It is poly-
Close the vitro cumulative burst size of object A drug-carrying nanometer particles, polymer B drug-carrying nanometer particle and polymer C drug-carrying nanometer particles same time
It reduces successively.The result is shown in Figure 11.
Hypoglycemic effect is investigated in 9 diabetic mice body of embodiment
The foundation of embodiment 9-1 models
It is (28 ± 5) g to take kunming mice, weight, and glycosuria is built using the low dose of streptozotocin of intraperitoneal injection in continuous more days
Sick mouse model, fasting 12 hours, dosage 30mgkg before being administered-1, continuous 2-3 weeks, the blood glucose of experiment mice is monitored,
Work as fasting blood-glucose>11.1mmol/L or non-fasting blood-glucoses>16.7mmol/L is considered as modeling success.
Hypoglycemic effect is investigated in embodiment 9-2 bodies
Diabetic mice is divided into 5 groups, it every group three, fasting 12h before being administered, can free water.A groups are diabetes model
Mouse non-administered group, that is, diabetic controls group；B groups are positive controls, inject commercially available insulin preparation solution 4U；Other three groups
It is diabetic mice administration group, PEI is subcutaneously injected in C groups10000-PCL4000-PEG2000-PCL4000-PEI10000Medicine is carried to receive
PEI is subcutaneously injected in grain of rice solution, D groups10000-PCL5000-PEG2000-PCL5000-PEI10000Drug-carrying nanometer particle solution, E groups are subcutaneous
Inject PEI10000-PCL6000-PEG2000-PCL6000-PEI10000Drug-carrying nanometer particle solution.Figure 12 time point tail veins are pressed after administration
It takes and detects blood glucose value with automatic blood glucose meter after blood.When carrying out statistical analysis, with SPSS20.0 statistical packages, surveyed using repetition
Variance analysis is measured, is compared two-by-two using LSD methods, P between group<0.05 is significant difference.
As a control group by A groups, the blood glucose level variation of C administration groups and the blood glucose level of A group diabetic mices change phase
Than there was no significant difference (P=0.226)；The blood glucose level variation of D administration groups and the blood glucose level of A group diabetic mices change
It compares, there is significant difference (P=0.007)；The blood glucose level of the blood glucose level variation and A group diabetic mices of E administration groups
Variation is compared, and has significant difference (P=0.017).Statistical result shows that the blood sugar decreasing effect of D, E administration group is apparent, D administrations
The blood sugar decreasing effect of group is better than E administration groups.
Using B groups as positive controls, commercially available insulin preparation is subcutaneously injected in blood glucose level variation and the B groups of C administration groups
Diabetic mice blood glucose level variation compare, no statistical discrepancy (P=0.945)；The blood glucose level of D administration groups changes and B
The blood glucose level variation of group mouse is compared, and has significant difference (P=0.045)；Blood glucose level variation and the B groups of E administration groups
The blood glucose level variation of mouse is compared, and there was no significant difference (P=0.117)；Statistical result shows that compared with B groups, C administration groups drop
Blood glucose effect lasts act on unobvious and D and E administration group hypoglycemic effect duration more commercially available insulin preparation is obviously prolonged,
Longest, that is, D groups can maintain the hypoglycemic time to be up to 34h, and subsequent blood glucose value is gradually increasing.As a result referring to Figure 12.
Reference
[1] Dong Anjie；Lin Xiaona；Deng Liandong by chemical bond carrying medicament temperature sensing in situ gel rubber and prepare application numbers：
CN201110437116
[2] soup morning sunlight；Zhao Li；Ding Jianxun；He Pan；Xiao Chunsheng；The village is beautiful；A kind of insulin drug carried microspheres of Chen Xuesi and
Preparation method application numbers：CN201110023708
[3] Zhu Limin；Remaining lamp is wide；Shen Xiaxia；Wu Chengyao；A kind of spontaneously-combined chitosan medicine-carrying nano particles of Nie Wei and its system
Preparation Method application numbers：CN201010132541
Claims (17)
1. a kind of pharmaceutical carrier, which is characterized in that it is made of five block copolymer PEI-PCL-PEG-PCL-PEI, and described five
Block copolymer PEI-PCL-PEG-PCL-PEI is PEIa-PCLb-PEGc-PCLd-PEIe, wherein a and e is respectively 10000, c
It is respectively 4000-6000 for 2000, b and d.
2. pharmaceutical carrier according to claim 1, the five block copolymers PEI-PCL-PEG-PCL-PEI are
PEI10000-PCL4000-PEG2000-PCL4000-PEI10000, PEI10000-PCL5000-PEG2000-PCL5000-PEI10000Or
PEI10000-PCL6000-PEG2000-PCL6000-PEI10000。
3. pharmaceutical carrier according to claim 1, the pharmaceutical carrier is by by the five block copolymers PEI-
PCL-PEG-PCL-PEI is dissolved with organic solution, is then added drop-wise in aqueous solution, is stirred and is formed nano-solution.
4. pharmaceutical carrier according to claim 1 or 2 includes macromolecular polypeptides class drug or micromolecular compound preparing
Drug in application, the macromolecular polypeptides be insulin.
5. according to the preparation method of claim 1-3 any one of them pharmaceutical carriers, it includes following steps：
1) synthesis triblock polymer PCLb-PEGc-PCLd
PCL is synthesized using ring-opening polymerisation methodb-PEGc-PCLdCopolymer, with PEGcIt is reacted with ε-CL, and with the ε-of 1%mol
CL amounts using stannous octoate as catalyst；
2) PCL of acrylic ester synthesizing sealing endb-PEGc-PCLd
By PCL obtained by step 1)b-PEGc-PCLdIt is dissolved with organic solvent, then is separately added into triethylamine and acryloyl chloride, molar ratio
For triethylamine:Acryloyl chloride:PCLb-PEGc-PCLd=4:4.2:1, stirring is to after reaction, being down to room temperature, mistake at 80 DEG C
Triethylamine hydrochloride is filtered out to crystallize；Filtered liquid precipitates in the n-hexane of excessive ice, and acrylate is collected after suction filtration
The PCL of sealing endb-PEGc-PCLd；
3) five block polymer PEI are synthesizeda-PCLb-PEGc-PCLd-PEIe
PEI is synthesized using Michael addition reactionsa-PCLb-PEGc-PCLd-PEIe, respectively by PEI and acrylate ended
PCLb-PEGc-PCLdBe dissolved in organic solvent respectively, by PEI chloroformic solutions under the conditions of 60 DEG C stirring and dissolving, then to PEI chloroforms
The PCL of acrylate ended is added dropwise in solutionb-PEGc-PCLdChloroformic solution vacuumizes logical nitrogen and continues afterwards in triplicate
It is stirred at reflux reaction for 24 hours, after reaction, is down to room temperature, crude product precipitates in excessive cold petroleum ether, and white wax is obtained by filtration
Shape precipitates, and after product is dissolved with q. s. methylene chloride, acquired solution instills in deionized water stirring dropwise, and to form milky molten
Liquid is stirred overnight freeze-drying acquisition PEI after volatile organic solventa-PCLb-PEGc-PCLd-PEIe。
6. a kind of slow release nano-particle, which is characterized in that comprising at least one active constituent and according to any one of claim 1-3
The pharmaceutical carrier.
7. slow release nano-particle according to claim 6, which is characterized in that the active constituent is macromolecular polypeptides class drug
Or micromolecular compound, the polypeptide drug are insulin.
8. the mass ratio (w/w) of slow release nano-particle according to claim 7, the insulin and pharmaceutical carrier is 10-
50%.
9. the mass ratio (w/w) of slow release nano-particle according to claim 7, the insulin and pharmaceutical carrier is 20-
40%.
10. the mass ratio (w/w) of slow release nano-particle according to claim 7, the insulin and pharmaceutical carrier be 20%,
25%, 30%, 35%, 40%.
11. according to claim 6-10 any one of them slow release nano-particle answering in the drug for preparing treatment metabolic disease
With the active constituent is insulin, and the metabolic disease is selected from diabetes.
12. according to claim 6-10 any one of them slow release nano-particle answering in the drug for preparing treatment metabolic disease
With the active constituent is insulin, and the metabolic disease is selected from hyperglycemia.
13. application according to claim 11, wherein diabetes are type-1 diabetes mellitus or type-2 diabetes mellitus.
14. according to the preparation method of claim 6-10 any one of them slow release nano-particles, include the following steps：
(1) active ingredient solution is prepared,
(2) pharmaceutical carrier is dissolved in organic solvent,
(3) active ingredient solution is added dropwise in pharmaceutical carrier solution, stirring, volatile organic solvent,
(4) it centrifuges and collects precipitation to get polymer drug-carried slow release nano-particle.
15. the preparation method of slow release nano-particle according to claim 14, the active constituent is insulin, including as follows
Step：
(1) precision weighs 4.0mg insulin, is dissolved in 0.01mol/L HCl solutions, then adjusts it with 1mol/L NaOH solutions
PH value obtains insulin solutions to 5.35 or more；
(2) pharmaceutical carrier is dissolved in dichloromethane organic solvent；
(3) insulin solutions are added dropwise to 10s/d rate of addition dropwise under magnetic stirring, be stirred at room temperature after 1~2h again 40
4h is persistently stirred under the conditions of DEG C, and organic solvent is made to volatilize naturally；
(4) 1h is centrifuged under the conditions of 14000rpm/min and collects precipitation, and is freeze-dried to get pancreas islet after being washed with deionized water
Plain slow release nano-particle.
16. the pH value in the preparation method of slow release nano-particle according to claim 15, wherein step (1) is adjusted to 5.8-
6.2。
17. preparation method according to claim 15, pH value are adjusted to 6.
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| CN104383533A (en) * | 2014-09-30 | 2015-03-04 | 温州医科大学 | Application of cationic polymer to prepare nanometer immunologic adjuvant and preparation method |
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