CN105646911B - A kind of liver target drug-carrying microballoon and its preparation method and application of pH and reduction responsiveness - Google Patents
A kind of liver target drug-carrying microballoon and its preparation method and application of pH and reduction responsiveness Download PDFInfo
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Abstract
Has the function of liver active targeting and with pH and the reduction microballoon of stimulating responsive, preparation method and purposes in medicine the present invention relates to a kind of.It is characterized in that: (1) vehicle group become galactose modified gelatin-poly- (beta-amino ester), microballoon contains linear poly- (the beta-amino ester) of disulfide bond using galactose modified gelatin, end group as secondary amine for monomer, using dialdehyde as crosslinking agent, prepared through emulsion-crosslinking method；(2) drug bearing microsphere has the constituent of antineoplastic and galactose modified gelatin-poly- (beta-amino ester), it is using doxorubicin hydrochloride as antineoplastic model drug, it is dispersed in monomer galactose modified gelatin and linear poly- (beta-amino ester), after crosslinking agent is added, two kinds of monomers crosslink reaction and are formed by microballoon；(3) microballoon prepared by has good evident characteristics to liver cell, has good pH and also primary stimuli release performance to model drug, can further develop to treat the novel targeted preparation of liver cancer.
Description
Technical field
The present invention relates to a kind of novel microballoons, and in particular to has the function of liver active targeting and has pH and reduction thorn
Swash the microballoon of responsiveness with and preparation method thereof and purposes medically, belong to high molecular material and biomedical engineering neck
Domain.
Technical background
Liver cancer has ranked China's tumour and has caused dead second, and chemotherapy is that mid and late liver cancer can not carry out liver transfer operation and liver
Important treatment means when excision.Often because of physicochemical property, poor, half-life short and effect cause anti-tumor drug without selection etc.
Therapeutic effect difference and serious toxicity.There is response to specific environment stimulation by embedding, assembling or being adsorbed on by drug
Property carrier inside, avoid the direct interaction of chemotherapeutics and normal tissue, make drug only site of action discharge, can mention
The selectivity of high drug, the toxic side effect for reducing drug.
For tumor tissues compared with normal tissue, the pH value in solid tumor is lower, weakly acidic, and the outer base of normal tissue cell
PH maintains 7.4 in matter and blood.In addition, the concentration of reductive glutathione (GSH) is 10~100 μM in blood, but swollen
Its concentration then rises to 1~10mM in oncocyte.Utilize difference of these of tumor tissues and normal tissue on pH and reproducibility
It is different, develop the drug-loading system that there is the anti-tumor drug of responsiveness to pH, also primary stimuli.
2013, Polymer Chemistry (Issue 4, pp1199-1207) was reported using atom transferred free radical
The polymerization reaction synthesis poly- N of mono methoxy polyethylene glycol-, N '-diethylaminoethyl acrylate copolymer (mPEG-b-PDEA),
MPEG-b-PDEA hydrogel is prepared for after quaternary ammoniated cross-linking reaction, using this hydrogel as the load of antineoplastic adriamycin
Body, keeps stable nucleocapsid structure in physiological conditions, and in acid and reproducibility tumor tissues and into the cell can be quick
Drug is released, thus realize the intelligence of drug release, but the carrier material does not have active targeting function, can not achieve
The targeting of antineoplastic conveys.
Summary of the invention
In view of this, having the function of liver active targeting technical problem to be solved by the invention is to provide one kind and having
PH and reduction stimulating responsive microballoon with and preparation method thereof, drug bearing microsphere can pass through targeting improve chemotherapeutic treatment effect
Fruit simultaneously mitigates adverse reaction caused by chemotherapeutic, drug resistance etc., can be used for the targeted therapy of liver cancer.
Has the function of liver active targeting and with pH and the galactolipin for restoring stimulating responsive the present invention provides a kind of
Poly- (beta-amino ester) microballoon (galG-PBAE microballoon) of modified gelatin-, it is characterized in that: having the galactolipin of the structure such as formula (I) to change
The constituent of property gelatin-poly- (beta-amino ester) (galG-PBAE), microballoon is by galactose modified gelatin (galG) monomer, end group
It is obtained for linear poly- (beta-amino ester) (PBAE) monomer that secondary amine contains disulfide bond by the emulsification and cross linked reaction of crosslinking agent.
The monomer galG, the structure with formula (II), passes through modified-reaction to gelatin (gel) by galactolipin (gal)
It obtains；
The monomer PBAE, the structure with formula (III), n is the degree of polymerization.By bis- (allyl acyloxyethyl) disulfides
(IV) it is obtained with N, N '-dimethyl hydrocarbon diamines (V) reaction.In formula (v), R1For-(CH2)2-、-(CH2)3-、-(CH2)4-、-
(CH2)5-、-(CH2)6-、 Or
The crosslinking agent is dialdehyde, the structure with formula (VI).In formula (VI), R2For no atom ,-CH2-、-
(CH2)2-、-(CH2)3-、-(CH2)4-、OrI.e. crosslinking agent used is glyoxal, malonaldehyde, fourth two
Aldehyde, glutaraldehyde, hexandial, terephthalaldehyde, m-terephthal aldehyde or o-phthalaldehyde.
The present invention also provides it is a kind of have the function of liver active targeting and with pH and restore stimulating responsive load medicine/
Galactose modified gelatin-poly- (beta-amino ester) microballoon (carry medicine/galG-PBAE microballoon), it is characterised in that: have antineoplastic and
The constituent of galG-PBAE is dispersed in antineoplastic in monomer galG and monomer PBAE, after crosslinking agent is added, two kinds
Monomer crosslinks reaction and is formed by microballoon.
The antineoplastic is doxorubicin hydrochloride (DOX) or other antineoplastics；
The monomer galG, the structure with formula (II), passes through modified-reaction to gelatin (gel) by galactolipin (gal)
It obtains；
The monomer PBAE, the structure with formula (III), n is the degree of polymerization.By bis- (allyl acyloxyethyl) disulfides
(IV) it is obtained with N, N '-dimethyl hydrocarbon diamines (V) reaction.In formula (V), R1For-(CH2)2-、-(CH2)3-、-(CH2)4-、-
(CH2)5-、-(CH2)6-、 Or
The crosslinking agent is dialdehyde, the structure with formula (VI).In formula (VI), R2For no atom ,-CH2-、-
(CH2)2-、-(CH2)3-、-(CH2)4-、OrI.e. crosslinking agent used is glyoxal, malonaldehyde, fourth two
Aldehyde, glutaraldehyde, hexandial, terephthalaldehyde, m-terephthal aldehyde or o-phthalaldehyde.
The present invention additionally provides simultaneously a kind of has the function of liver active targeting and with pH and reduction stimulating responsive
The preparation method of galG-PBAE microballoon, it is characterized in that: using galG and PBAE as monomer, using dialdehyde as crosslinking agent, through emulsification and cross linked
Method preparation.The specific steps of preparation are as follows:
(1) monomer galG and monomer PBAE are proportionally added into water, after being dispersed with stirring uniformly, water phase is made.
The mass ratio of monomer galG and monomer PBAE are (1~2): (1~2), monomer galG and monomer PBAE are dispersed in water
In total volume concentration be 25~200mg/ml；
(2) emulsifier is dispersed in atoleine in proportion, oil phase is made.
Emulsifier used be span 80 or tween 80, volumetric concentration of the emulsifier in atoleine be 0.5%~
5%；
(3) under conditions of temperature is 25~85 DEG C and mixing speed is 50~1500rpm, water phase is added dropwise to oily phase.
Oil phase volume used is 5~50 times of water phase volume；
(4) under conditions of temperature is -5 DEG C~10 DEG C and mixing speed is 50~1500rpm, crosslinking agent is added and is handed over
Connection reaction.
Crosslinking agent used is glyoxal, malonaldehyde, butanedial, glutaraldehyde, hexandial, terephthalaldehyde, m-terephthal aldehyde
Or o-phthalaldehyde, dosage are 0.5~5 times of both galG and PBAE gross mass；
(5) solid content is collected, after isopropanol, normal propyl alcohol, ethyl alcohol, methanol or acetone washing, then with petroleum ether, very
Sky is dry, obtains galG-PBAE microballoon.
The present invention, which still further provides, a kind of to be had the function of liver active targeting and with pH and restores stimulating responsive
The preparation method of drug bearing microsphere, characterized in that using galG and PBAE as monomer, using dialdehyde as crosslinking agent, using DOX as antineoplastic
Model drug prepares through emulsion-crosslinking method and carries medicine/galG-PBAE microballoon.
Load medicine/galG-PBAE microballoon preparation method, includes the following steps:
(1) monomer galG and monomer PBAE are proportionally added into water, after being dispersed with stirring, are proportionally added into antitumor model
Drug DOX, stirs evenly, and water phase is made.
The mass ratio of monomer galG and monomer PBAE are (1~2): (1~2), the gross mass of both monomer galG and PBAE
For 5~50 times of antitumor model drug DOX mass, the total volume concentration that monomer galG and monomer PBAE are dispersed in water is 25
~200mg/ml；
(2) emulsifier is dispersed in atoleine in proportion, oil phase is made.
Emulsifier used be span 80 or tween 80, volumetric concentration of the emulsifier in atoleine be 0.5%~
5%；
(3) under conditions of temperature is 25~85 DEG C and mixing speed is 50~1500rpm, water phase is added dropwise to oily phase.
Oil phase volume used is 5~50 times of water phase volume；
(4) under conditions of temperature is -5 DEG C~10 DEG C and mixing speed is 50~1500rpm, crosslinking agent is added and is handed over
Connection reaction.
Crosslinking agent used is glyoxal, malonaldehyde, butanedial, glutaraldehyde, hexandial, terephthalaldehyde, m-terephthal aldehyde
Or o-phthalaldehyde, dosage are 0.5~5 times of both galG and PBAE gross mass；
(5) solid content is collected, after isopropanol, normal propyl alcohol, ethyl alcohol, methanol or acetone washing, then with petroleum ether, very
Sky is dry, obtains carrying DOX/galG-PBAE microballoon.
The preparation method of the monomer galG (II), it is anti-through O- acetylation protection and bromo by β-D- galactolipin (IIa)
1- bromo -2,3 should be synthesized, obtains 2,3,4,6- tetrems after 4,6- tetra- acetoxyl group-β-D- galactolipins (IIb), with thiocarbamide condensation
Thio-β-D- galactosyl-the 2 '-isothiourea hydrobromide (IIc) of acyloxy -1-, reacts in the presence of acid binding agent with chloroacetonitrile
Obtain 2,3,4,6- tetra- acetoxyl group -1- nitrile methyl thio-β-D- galactolipins (IId), in the presence of sodium methoxide with carbinol condensation
Thio-β-D- the galactolipin (IIe) of 2- imino group -2- methoxyethyl -1- is obtained, the latter and gelatin are condensed to yield galG.It is specific anti-
Answer step are as follows:
The preparation method of the monomer PBAE (III), by bis- (2- ethoxy) disulfides (IIIa) at triethylamine (TEA)
In the presence of react to obtain bis- (allyl acyloxyethyl) disulfides (IIIc), the latter and N, N '-dimethyl with acryloyl chloride (IIIb)
Hydrocarbon diamines obtains PBAE through Michael's addition polymerization reaction.Specific reaction step are as follows:
Compared with prior art, the present invention having the advantage that
(1) the beta-amino ester bond of the Schiff base of the crosslinked formation of microsphere surface and linear poly- (beta-amino ester) intramolecular is equal
It is hydrolyzed under conditions of acid lower faster, the drug bearing microsphere of preparation is made to keep structural integrity in the normal tissue, and in acidity
Rapid cleavage discharges drug in lower tumor tissues, and this structure makes the drug bearing microsphere of preparation have pH stimulating responsive；
In addition, the disulfide bond sensitive to reproducibility is introduced in linear poly- (beta-amino ester), so that the drug bearing microsphere of preparation is normal
Complete structure is kept in tissue, and rapid cleavage discharges drug in the tumor tissues of reproducibility, this structure makes preparation
Microballoon is simultaneously but also with reduction stimulating responsive；
(2) microsphere surface introduces galactosyl ligand, and the identification which is capable of selectivity is only expressed in mammal
The asialoglycoprotein receptor (ASGR) of surface of hepatocytes adheres to microballoon selectively by ligand-receptor interaction
In surface of hepatocytes, active liver targeting function is shown.This hepatic targeting is gathered in microballoon selectively in hepatic tissue,
Rapid delivery of pharmaceuticals under cancerization region pH, reduction double stimuli, makes liver cancer cells be infiltrated on the chemotherapeutics of high concentration for a long time
In, drug effect is enhanced, adverse reaction, drug resistance etc. are alleviated；
(3) it is also an advantage of the present invention that raw material is cheap and easy to get, preparation process is simple, preparation condition requires low, encapsulation rate
High, microspherulite diameter is suitable for that it is as drug delivery body that gelatin used and poly- (beta-amino ester), which have good biocompatibility,
The preferred material of system.
Detailed description of the invention
Prepared load DOX/galG-PBAE microballoon is in pH5.5, pH 7.4, pH 5.5 and DTT in Fig. 1 embodiment 1
The relationship of cumulative release amount at any time in 10mM solution
It prepared loads DOX/galG-PBAE microballoon and the HepG2 cell for expressing ASGR and is not expressed in Fig. 2 embodiment 1
The adherency inverted fluorescence microscope photo of the Hela cell of ASGR, wherein (a) is that HepG2 cell and load DOX/galG-PBAE are micro-
Ball is (b) Hela cell and load DOX/galG-PBAE microballoon
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.
Embodiment 1
(1) galactose modified gelatin (II) is prepared
1- bromo -2,3, the preparation of 4,6- tetra- acetoxyl group-β-D- galactolipins (IIb): 16ml acetic anhydride is under 0 DEG C of stirring
0.08ml HClO is added4, it is then slowly added to 4g β-D- galactolipin (IIa) in 30min, is warming up to 35~40 DEG C of reactions
30min.1.2g red phosphorus, 2.3ml Br are sequentially added under ice cooling, 42With 1.5ml water, temperature is increased to 18~20 DEG C of reactions
2h.20ml chloroform is added, is poured into ice water after stirring.Filtering, liquid separation, water phase are extracted twice with 20ml chloroform, merge chloroform, are used
It is saturated NaHCO3, distillation water washing to water phase be neutrality.Anhydrous MgSO is added4It is dried overnight, vacuum rotary steam removes chloroform and obtains Huang
Sugar colour starches sample object.With ether dissolution and petroleum ether is added until when the white precipitate generated is not redissolved just, is placed in refrigerator
Middle crystallization, obtains white crystal IIb, and fusing point is 78~80 DEG C.
The preparation of thio-β-D- galactosyl-the 2 '-isothiourea hydrobromide (IIc) of 2,3,4,6- tetra- acetoxyl group -1-:
2.79g IIb and 0.515g thiocarbamide is added in advance added with 5ml anhydrous propanone and in the drying three-neck flask with reflux unit,
60 DEG C of reflux 30min, slightly cold be placed in refrigerator crystallize.White crystal IIc is obtained, fusing point is 168~170 DEG C.
The preparation of 2,3,4,6- tetra- acetoxyl group -1- nitrile methyl thio-β-D- galactolipins (IId): the IIb and 1ml of 1.9g
Chloroacetonitrile is added to the in the mixed solvent of acetone and water that 10ml volume ratio is 1: 1, and stirring to solid is added after all dissolving
0.8g K2CO3With 1g NaHSO3, continue to stir 30min at room temperature.Then 70ml ice water is added and is vigorously stirred 2h.Filtering
After collecting solid, vacuum drying, IId is obtained through 10ml recrystallizing methanol, fusing point is 95~97 DEG C.
The preparation of the thio-β-D- galactolipin (IIe) of 2- imino group -2- methoxyethyl -1-: 1.209g IIc and 0.016g first
Sodium alkoxide is added in the anhydrous methanol of 30ml, reacts 9h at 37 DEG C.Vacuum rotary steam removes methanol and obtains yellow syrup object (IId).
The preparation of galactose modified gelatin (II): 0.5g gelatin is added in 50ml borate buffer solution (pH 8.0) and is made into
Gelatin solution is added 0.5g IId, reacts at room temperature for 24 hours.Vinegar acid for adjusting pH is added to 4~5 quenching reactions, then uses semi-permeable membrane
Dialyse 48h, and freeze-drying obtains galactose modified gelatin (II).
(2) preparing end group is linear poly- (beta-amino ester) (III) that secondary amine contains disulfide bond
The preparation of bis- (allyl acyloxyethyl) disulfides (IIIa): bis- (2- ethoxy) disulfides of 0.60g are anhydrous with 15ml
Tetrahydrofuran dissolution, is cooled to -10 DEG C, and 2.2ml triethylamine is added and stirs 10min.Be added dropwise 1.45g acryloyl chloride be dissolved in 5ml without
The solution of water tetrahydrofuran is to slowly warm up to 20 DEG C the reaction was continued 12h.It is filtered to remove the solid of generation after reaction, depressurizes
Revolving removes tetrahydrofuran, obtained yellow viscous liquid CHCl3Dissolution, respectively with saturation Na2CO3Solution and distillation washing
It washs to neutrality.CHCl3Layer is through anhydrous MgSO4It after being dried overnight, filters, revolving, crude product separates (eluant, eluent through silica gel column chromatography
For ethyl acetate: n-hexane=1: 3 mixed solvent), obtain weak yellow liquid IIIa.
End group is the preparation for the linear poly- beta-amino ester (III) that secondary amine contains disulfide bond: 115mg IIIa is dissolved in
10ml anhydrous tetrahydro furan, in N2Protection is lower to be added 52.8mgN, and N '-dimethyl ethylenediamine is warming up to 50 DEG C and is protected from light 48h.
Revolving removes tetrahydrofuran, and residue is slowly added dropwise into the cold anhydrous ether being vigorously stirred, and precipitation obtains faint yellow sticky
Object III.
(3) microballoon is prepared
The preparation of unloaded microballoon:
40.5mg mass ratio is dissolved in 1ml distillation for 1: 1 galactose modified gelatin (II) and poly- beta-amino ester (III)
Water.With saturation Na2CO3Solution adjusts pH to 9, obtains water phase.
0.15ml span 80 is added in 15ml atoleine, 30min is stirred in 50 DEG C and revolving speed 600rpm, obtains
Oily phase.
Under the mixing speed of 600rpm, water phase is added dropwise in oily phase.It is cooled to 0 DEG C, is added 0.5g's 25%
Glutaraldehyde is protected from light 2h at 0 DEG C.Then 15ml isopropanol is added, continues to stir 30min, filtering, solid content is through isopropyl
Alcohol obtains unloaded microballoon after petroleum ether 3~4 times.
Gained zero load microballoon is spherical shape, and good dispersion, average grain diameter is 7.9 μm, to human liver cancer cell HepG2 and people uterus
Two class of oncocyte Hela is intercellular to be stained with highly selective, can be had by the galactosyl specific recognition ASGR on surface
There is active liver targeting function.
The preparation of drug bearing microsphere:
In addition to 4.5mg doxorubicin hydrochloride is added when water is mutually prepared, remaining presses the preparation method of unloaded microballoon in the present embodiment.
The process for preparation of the water phase are as follows: 40.5mg mass ratio is molten for 1: 1 galactose modified gelatin (II) and poly- beta-amino ester (III)
In 1ml distilled water, 4.5mg doxorubicin hydrochloride is added, stirs evenly.With saturation Na2CO3Solution adjusts pH to 9, obtains water phase.
Gained drug bearing microsphere is spherical shape, and good dispersion, average grain diameter is 7.0 μm；Drugloading rate is 6.61%, and encapsulation rate is
66.08%.Load DOX/galG-PBAE microballoon is reducing pH or is increasing the concentration Shi Junneng of reducing agent dithiothreitol (DTT)
Rate of release is accelerated when accelerating the release of drug, while reducing pH and increasing the concentration of DTT and drug release amount dramatically increases (see figure
1)；It is strong to the adhesive capacity of human liver cancer cell HepG2, and it is weak (see Fig. 2) to the adhesive capacity of people uterus oncocyte Hela cell,
Illustrate that the load DOX/galG-PBAE microballoon of preparation there can be active liver by the galactosyl specific recognition ASGR on surface
Target function.
Embodiment 2
Galactose modified gelatin is prepared as described in Example 1.
As described in Example 1 with 69.6mg N, N '-dimethyl butanediamine replaces N, N '-dimethyl ethylenediamine, preparation two
End is in secondary amine repetitive unit linear poly- (beta-amino ester) containing disulfide bond.
The preparation of unloaded microballoon:
45mg mass ratio is dissolved in 1ml distilled water for 2: 1 galactose modified gelatin and poly- beta-amino ester.With saturation
Na2CO3Solution adjusts pH to 9, obtains water phase.
0.25ml span 80 is added in 50ml atoleine, 30min is stirred at 85 DEG C and revolving speed 550rpm, obtains
Oily phase.
Under the mixing speed of 550rpm, water phase is added dropwise in oily phase.Continue after stirring 20min, is cooled to -5
DEG C, the glutaraldehyde of 0.1g 25% is added, is protected from light 2h at -5 DEG C.Then 15ml ethyl alcohol is added, continues to stir 30min, mistake
Filter, solid content obtain unloaded microballoon after ethyl alcohol, petroleum ether 3~4 times.
Gained zero load microballoon is spherical shape, and good dispersion, average grain diameter is 26.1 μm, to two class iuntercellular of HepG2 and Hela
Be stained with it is highly selective, can by the galactosyl specific recognition ASGR on surface have active liver targeting function.
The preparation of drug bearing microsphere:
In addition to 9mg doxorubicin hydrochloride is added when water is mutually prepared, remaining presses the preparation method of unloaded microballoon in the present embodiment.
Gained drug bearing microsphere is spherical shape, and good dispersion, average grain diameter is 27.4 μm；Drugloading rate is 10.38%, and encapsulation rate is
63.78%.The drug bearing microsphere releases the drug in the environment of pH 5.5 and 10mM DTT, for 24 hours cumulative release amount of drug 78.36%；It is right
HepG2 and two class of Hela is intercellular is stained with highly selective, can pass through the galactosyl specific recognition ASGR on surface
With active liver targeting function.
Embodiment 3
Galactose modified gelatin is prepared as described in Example 1.
As described in Example 1 with 86.4mg N, N '-dimethyl hexamethylene diamine replaces N, N '-dimethyl ethylenediamine, preparation two
End is in secondary amine repetitive unit linear poly- (beta-amino ester) containing disulfide bond.
The preparation of unloaded microballoon:
50mg mass ratio is dissolved in 2ml distilled water for 1: 2 galactose modified gelatin and poly- beta-amino ester.With saturation
Na2CO3Solution adjusts pH to 9, obtains water phase.
0.5ml span 80 is added in 10ml atoleine, 30min is stirred at 25 DEG C and revolving speed 50rpm, obtains oil
Phase.
Under the mixing speed of 50rpm, water phase is slowly added into dropwise in oily phase.Continue after stirring 20min, is cooled to
5 DEG C, the glyoxal of 62.5mg40% is added, is protected from light 2h at 5 DEG C.Then 15ml methanol is added, continues to stir 30min,
Filtering, solid content obtain unloaded microballoon after methanol, petroleum ether 3~4 times.
Gained zero load microballoon is spherical shape, and good dispersion, average grain diameter is 56.0 μm, to two class iuntercellular of HepG2 and Hela
Be stained with it is highly selective, can by the galactosyl specific recognition ASGR on surface have active liver targeting function.
The preparation of drug bearing microsphere:
In addition to 1mg doxorubicin hydrochloride is added when water is mutually prepared, remaining presses the preparation method of unloaded microballoon in the present embodiment.
Gained drug bearing microsphere is spherical shape, and good dispersion, average grain diameter is 54.5 μm；Drugloading rate is 1.46%, and encapsulation rate is
61.78%.The drug bearing microsphere releases the drug in the environment of pH 5.5 and 10mM DTT, for 24 hours cumulative release amount of drug 75.62%；It is right
HepG2 and two class of Hela is intercellular is stained with highly selective, can pass through the galactosyl specific recognition ASGR on surface
With active liver targeting function.
Embodiment 4
Galactose modified gelatin is prepared as described in Example 1.
As described in Example 1 with 81.6mg N, N '-dimethyl p-phenylenediamine replaces N, N '-dimethyl ethylenediamine, preparation
Both ends are in secondary amine repetitive unit linear poly- (beta-amino ester) containing disulfide bond.
The preparation of unloaded microballoon:
100mg mass ratio is dissolved in 0.5ml distilled water for 1: 1 galactose modified gelatin and poly- beta-amino ester.With saturation
Na2CO3Solution adjusts pH to 9, obtains water phase.
1ml tween 80 is added in 25ml atoleine, 30min is stirred in 50 DEG C and revolving speed 1500rpm, obtains
Oily phase.
Under the mixing speed of 1500rpm, water phase is added dropwise in oily phase.Continue after stirring 20min, is cooled to 10
DEG C, 0.1g terephthalaldehyde is added, is protected from light 2h at 10 DEG C.Then 30ml acetone is added, continues to stir 30min, filter,
Filter residue obtains unloaded microballoon after acetone, petroleum ether 3~4 times.
Gained zero load microballoon is spherical shape, and good dispersion, average grain diameter is 19.5 μm, to two class iuntercellular of HepG2 and Hela
Be stained with highly selective, can have by the galactosyl specific recognition ASGR on surface and actively swell target function.
The preparation of drug bearing microsphere:
In addition to 10mg doxorubicin hydrochloride is added when water is mutually prepared, remaining presses the preparation method of unloaded microballoon in the present embodiment.
Gained drug bearing microsphere is spherical shape, and good dispersion, average grain diameter is 18.2 μm；Drugloading rate is 6.18%, and encapsulation rate is
65.12%.The drug bearing microsphere releases the drug in the environment of pH 5.5 and 10mM DTT, for 24 hours cumulative release amount of drug 80.12%；It is right
HepG2 and two class of Hela is intercellular is stained with highly selective, can pass through the galactosyl specific recognition ASGR on surface
With active liver targeting function.
Claims (10)
1. a kind of have the function of liver active targeting and the microballoon with pH and reduction stimulating responsive, it is characterised in that: have
Such as the constituent of the galactose modified gelatin-of the structure of formula (I) poly- (beta-amino ester), be by monomer galactose modified gelatin,
Monomer end group, which reacts for linear poly- (beta-amino ester) that secondary amine contains disulfide bond with crosslinking agent, is formed by microballoon；
The monomer galactose modified gelatin, the structure with formula (II), by galactolipin (gal) to gelatin (gel) by changing
Property reacts to obtain；
The monomer end group is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond, has the structure of formula (III), and n is
The degree of polymerization, (allyl acyloxyethyl) disulfides bis- as shown in formula (IV) and N shown in formula (V), N '-dimethyl hydrocarbon diamine reactant
It obtains；In formula (V), R1For-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、
The crosslinking agent is dialdehyde, the structure with formula (VI), in formula (VI), R2For no atom ,-CH2-、-(CH2)2-、-
(CH2)3-、-(CH2)4-、
2. a kind of have the function of liver active targeting and the drug bearing microsphere with pH and reduction stimulating responsive, it is characterised in that:
Constituent with antineoplastic and galactose modified gelatin-poly- (beta-amino ester) is that antineoplastic is dispersed in monomer half
Lactose modification gelatin and monomer end group are that secondary amine contains in linear poly- (the beta-amino ester) of disulfide bond, after crosslinking agent is added, two
Kind monomer crosslinks reaction and is formed by drug bearing microsphere；
The antineoplastic is doxorubicin hydrochloride or other antineoplastics；
The monomer galactose modified gelatin, the structure with formula (II), by galactolipin (gal) to gelatin (gel) by changing
Property reacts to obtain；
The monomer end group is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond, has the structure of formula (III), and n is
The degree of polymerization, (allyl acyloxyethyl) disulfides bis- as shown in formula (IV) and N shown in formula (V), N '-dimethyl hydrocarbon diamine reactant
It obtains；In formula (V), R1For-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、
The crosslinking agent is dialdehyde, the structure with formula (VI), in formula (VI), R2For no atom ,-CH2-、-(CH2)2-、-
(CH2)3-、-(CH2)4-、
3. a kind of preparation method of microballoon as described in claim 1, it is characterised in that: with galactose modified gelatin and end group be secondary
Linear poly- (the beta-amino ester) that amido contains disulfide bond is that monomer is prepared using dialdehyde as crosslinking agent through emulsion-crosslinking method, preparation
Specific step is as follows:
(1) by monomer galactose modified gelatin and monomer end group be linear poly- (beta-amino ester) that secondary amine contains disulfide bond press than
Example is added to the water, and after being dispersed with stirring uniformly, water phase is made；
Monomer galactose modified gelatin and monomer end group are the mass ratio of linear poly- (beta-amino ester) that secondary amine contains disulfide bond
It is the linear poly- (beta-amino that secondary amine contains disulfide bond for (1~2): (1~2), monomer galactose modified gelatin and monomer end group
Ester) total volume concentration that is dispersed in water is 25~200mg/ml；
(2) emulsifier is dispersed in atoleine in proportion, oil phase is made；
Emulsifier used is span 80 or tween 80, and volumetric concentration of the emulsifier in atoleine is 0.5%~5%；
(3) under conditions of temperature is 25~85 DEG C and mixing speed is 50~1500rpm, water phase is added dropwise to oily phase；
Oil phase volume used is 5~50 times of water phase volume；
(4) under conditions of temperature is -5 DEG C~10 DEG C and mixing speed is 50~1500rpm, crosslinking agent is added and be crosslinked instead
It answers；
Dosage of crosslinking agent used is galactose modified gelatin and end group is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond
0.5~5 times of the two gross mass；
(5) collect solid content, after isopropanol, normal propyl alcohol, ethyl alcohol, methanol or acetone washing, then use petroleum ether, vacuum do
It is dry, obtain microballoon.
4. the preparation method of microballoon according to claim 3, it is characterised in that: monomer galactose modified gelatin (II) used
Preparation method is to synthesize 1- bromo -2,3,4,6- tetra- acetyl through O- acetylation protection and bromo-reaction by β-D- galactolipin (IIa)
2,3,4, the 6- tetra- thio-β-D- galactosyls-of acetoxyl group -1- are obtained after oxygroup-β-D- galactolipin (IIb), with thiocarbamide condensation
2 '-isothiourea hydrobromides (IIc) react to obtain 2,3,4,6- tetra- acetoxyl group -1- nitriles with chloroacetonitrile in the presence of acid binding agent
Methyl thio-β-D- galactolipin (IId) obtains 2- imino group -2- methoxyethyl -1- sulphur in the presence of sodium methoxide with carbinol condensation
Generation-β-D- galactolipin (IIe), the latter and gelatin are condensed to yield monomer galactose modified gelatin, specific reaction step are as follows:
5. the preparation method of microballoon according to claim 3, it is characterised in that: monomer end group used is that secondary amine contains two sulphur
The preparation method of linear poly- (beta-amino ester) (III) of key is to be existed by bis- (2- ethoxy) disulfides (IIIa) in triethylamine
It is lower to react to obtain bis- (allyl acyloxyethyl) disulfides (IIIc), the latter and N, N '-dimethyl hydrocarbon two with acryloyl chloride (IIIb)
It is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond that amine, which obtains monomer end group through Michael's addition polymerization reaction, specific anti-
Answer step are as follows:
6. a kind of preparation method of drug bearing microsphere as claimed in claim 2, it is characterised in that: with galactose modified gelatin and end group
Linear poly- (the beta-amino ester) for containing disulfide bond for secondary amine is monomer, using dialdehyde as crosslinking agent, with antineoplastic hydrochloric acid Ah mould
Element is model drug, is prepared through emulsion-crosslinking method, specific step is as follows for preparation:
(1) by monomer galactose modified gelatin and monomer end group be linear poly- (beta-amino ester) that secondary amine contains disulfide bond press than
Example is added to the water, and after being dispersed with stirring, is proportionally added into antitumor model drug doxorubicin hydrochloride, stirs evenly, and water phase is made；
Monomer galactose modified gelatin and monomer end group are the mass ratio of linear poly- (beta-amino ester) that secondary amine contains disulfide bond
It is the linear poly- (beta-amino that secondary amine contains disulfide bond for (1~2): (1~2), monomer galactose modified gelatin and monomer end group
Ester) both gross mass be 5~50 times of antitumor model drug doxorubicin hydrochloride quality, monomer galactose modified gelatin and list
Body end base is that the total volume concentration that linear poly- (the beta-amino ester) that secondary amine contains disulfide bond is dispersed in water is 25~200mg/
ml；
(2) emulsifier is dispersed in atoleine in proportion, oil phase is made；
Emulsifier used is span 80 or tween 80, and volumetric concentration of the emulsifier in atoleine is 0.5%~5%；
(3) under conditions of temperature is 25~85 DEG C and mixing speed is 50~1500rpm, water phase is added dropwise to oily phase；
Oil phase volume used is 5~50 times of water phase volume；
(4) under conditions of temperature is -5 DEG C~10 DEG C and mixing speed is 50~1500rpm, crosslinking agent is added and be crosslinked instead
It answers；
Dosage of crosslinking agent used is galactose modified gelatin and end group is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond
0.5~5 times of the two gross mass；
(5) collect solid content, after isopropanol, normal propyl alcohol, ethyl alcohol, methanol or acetone washing, then use petroleum ether, vacuum do
It is dry, obtain drug bearing microsphere.
7. the preparation method of drug bearing microsphere according to claim 6, it is characterised in that: monomer galactose modified gelatin used
(II) preparation method is, synthesizes 1- bromo -2,3 through O- acetylation protection and bromo-reaction by β-D- galactolipin (IIa), 4,6-
2,3,4, the 6- tetra- thio-β-D- galas of acetoxyl group -1- are obtained after four acetoxyl group-β-D- galactolipins (IIb), with thiocarbamide condensation
Glycosyl -2 '-isothiourea hydrobromide (IIc) reacts to obtain 2,3,4,6- tetra- acetoxyl groups-with chloroacetonitrile in the presence of acid binding agent
1- nitrile methyl thio-β-D- galactolipin (IId) obtains 2- imino group -2- methoxyethyl-in the presence of sodium methoxide with carbinol condensation
Thio-β-D- the galactolipin (IIe) of 1-, the latter and gelatin are condensed to yield monomer galactose modified gelatin, specific reaction step are as follows:
8. the preparation method of drug bearing microsphere according to claim 6, it is characterised in that: monomer end group used contains for secondary amine
The preparation method of linear poly- (beta-amino ester) (III) of disulfide bond is, by bis- (2- ethoxy) disulfides (IIIa) in triethylamine
In the presence of react to obtain bis- (allyl acyloxyethyl) disulfides (IIIc), the latter and N, N '-dimethyl with acryloyl chloride (IIIb)
It is linear poly- (the beta-amino ester) that secondary amine contains disulfide bond, tool that hydrocarbon diamines, which obtains monomer end group through Michael's addition polymerization reaction,
Precursor reactant step are as follows:
9. microballoon described in claim 1 is preparing pH and is restoring the anti-tumor drug load of double stimulating responsives and hepatic targeting
The application of body.
10. drug bearing microsphere as claimed in claim 2 is in the application of preparation treatment liver-cancer medicine.
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