CN102580106A - Method for preparing pH-sensitive type polyelectrolyte microcapsule administration carrier - Google Patents
Method for preparing pH-sensitive type polyelectrolyte microcapsule administration carrier Download PDFInfo
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Abstract
The invention discloses a method for preparing a pH-sensitive type polyelectrolyte microcapsule administration carrier. The method comprises the following steps of: (1) adding albumin into a calcium chloride aqueous solution and a sodium carbonate aqueous solution, and stirring uniformly to obtain albumin calcium carbonate particles; (2) dispersing the albumin calcium carbonate particles in a polyelectrolyte solution A, incubating at room temperature for 10 to 20 minutes, centrifuging, washing precipitates, dispersing in a polyelectrolyte solution B, incubating at room temperature for 10 to 20 minutes, centrifuging, performing primary double-layer coating, and coating repeatedly for 3 to 5 times to obtain colloid particles with core-shell structures; and (3) dissolving and decoring the colloid particles with the core-shell structures by using 0.2 mol/L of ethylene diamine tetraacetic acid (EDTA) solution, heating at the temperature of between 70 and 80 DEG C, so that albumin molecules are gelated fully to obtain pH-sensitive type polyelectrolyte microcapsules. By the method, the loading and release of the microcapsules on medicines can be controlled by regulating the pH value to achieve an intelligentized effect. The method is reasonable in design and has bright study and application prospects, and a preparation process is simple.
Description
(1) technical field
The present invention relates to a kind of method for preparing of pH responsive type drug administration carrier, relate in particular to a kind of method for preparing that is pre-charged with albuminous polyelectrolyte microcapsule drug administration carrier.
(2) background technology
Continuous development along with field of medicaments; In order to improve bioavailability of medicament, the toxic and side effects that reduces medicine and the maximization that obtains curative effect of medication, the multi-functional drug carriers that possesses multiple character such as long circulation, targeting and environment sensitive has simultaneously become one of pharmaceutics hot research fields in recent years.Environment sensitive is as an important ingredient, and medicine is encapsulated in can protect medicine in blood circulation, not to be degraded in the examples of such carriers, and discharges at specific position.
(Layer-by-layer is that two kinds of polyelectrolyte capsule materials with opposite charges alternately are adsorbed on the parent nucleus LBL), reaches to select after the desirable number of plies The suitable solvent to remove parent nucleus, obtains a kind of method of the polyelectrolyte microcapsule of hollow in self assembly layer by layer.Polyelectrolyte microcapsule based on layer-by-layer has caused concern widely in a plurality of fields such as drug delivery, catalysis, sensings.As drug carrier system; This microcapsule is except can control by size, composition, structure, form and the wall thickness to microcapsule on nanoscale accurately; And outside the preparation condition gentleness, its significant superiority also is the multi-functional of it and to the sensitivity of environment.Thereby pH, temperature, ionic strength, light etc. are the release of the permeability control medicine of its cyst wall of scalable all.But as pharmaceutical carrier, its drug loading is lower, and this has greatly influenced its development prospect as pharmaceutical carrier.The auto-deposition effect be meant drug molecule through with the electrostatic attraction effect of the polymer that is pre-filled in polyelectrolyte microcapsule the inside by high concentration be enriched in microcapsule inside; Thereby greatly improve drug loading, showing special advantages aspect the medicine carrying of polyelectrolyte microcapsule.But how controlling this auto-deposition effect as required and how discharging these medicines that tightly attracted by static is a challenge and meaning and the work of depositing.
Albumin is considered to the well carrier of natural fat-soluble medicine of a kind of biocompatibility, has been widely used in drug delivery system, like the paclitaxel albumin nano granular.Bovine serum albumin (BSA) is a kind of originate abundant and cheap albumin, and its molecular weight is about 66kD, is heated to about 70 ℃ gelation can take place.The isoelectric point, IP of BSA is 4.8, and BSA greater than 4.8 time is electronegative when pH value, and less than 4.8 o'clock BSA positively chargeds.This characteristic provides foundation for it is applied to pH responsive type carrier.BSA is pre-filled in the polyelectrolyte microcapsule, changes the electric charge of BSA again through the adjustment pH value, not only can the ground enrichment of medicine high concentration is inner to microcapsule, but also can control the release of medicine.
(3) summary of the invention
The present invention seeks to deficiency, a kind of method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier is provided to polyelectrolyte microcapsule present Research.
The technical scheme that the present invention adopts is:
A kind of method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier, said method is: (1) adds albumin that to make albuminous concentration in 0.025mol/L～0.33mol/L calcium chloride water be 0.5mg/mL～5mg/mL, stirring and dissolving; Under agitation add and the isopyknic aqueous sodium carbonate of described calcium chloride water same molar ratio rapidly; Continue to stir, leave standstill, get deposition; Drying obtains the albumin calcium carbonate microparticle; (2) getting albumin calcium carbonate microparticle that step (1) obtains operates as follows and 1. is dispersed in 0.5mg/mL～2mg/mL (in preferred 1～2mg/mL) the polyelectrolyte A solution; Room temperature (20 ℃) stirs hatches 10～20min; Centrifugalize; Get washing of precipitate, the quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL (being the albumin calcium carbonate microparticle that disperses 100～200mg in every 1mL polyelectrolyte A solution, preferred 100mg/mL) with the volume of polyelectrolyte A solution; 2. the precipitation after will washing again be dispersed in 0.5mg/mL～2mg/mL (preferred 1～2mg/mL) with the isopyknic polyelectrolyte B of polyelectrolyte A solution solution in; Room temperature (20 ℃) stirs and hatches 10～20min, and washing of precipitate is got in centrifugalize; Get the albumin calcium carbonate microparticle that deposition after the washing promptly obtains accomplishing 1 double-layer coatings; 3. with the albumin calcium carbonate microparticle repeatable operation behind 1 coating 1. with 2. step 3～5 time, accomplish repeatedly double-layer coatings, centrifugal; Washing, the colloidal particles of acquisition nucleocapsid structure; Said polyelectrolyte A and polyelectrolyte B are two kinds of polyelectrolyte with opposite charges; The colloidal particles of the nucleocapsid structure that (3) step (2) is obtained dissolve enucleation with the EDTA solution of 0.2mol/L; Centrifugal, to get deposition and be scattered in the deionized water, 70～80 ℃ of heating make the abundant gelation of albumin molecule; Centrifugal, deposition is said pH responsive type polyelectrolyte microcapsule drug administration carrier.
The said albumin of step (1) is bovine serum albumin or human serum albumin, excellent bovine serum albumin choosing.
The concentration of the said albumin of step (1) in 0.025mol/L～0.33mol/L calcium chloride water is 2.0mg/mL～3.0mg/mL.
Step (1) is got intermediate processing: reaction with the centrifugal 5～10min of reactant liquor 4000～7000r/min, is got deposition after finishing, and vacuum drying 48～72h obtains the albumin calcium carbonate microparticle.
Polyelectrolyte A according to the invention and B can be various polyelectrolyte with opposite charges, are preferably PAH hydrochlorate, kayexalate respectively; Chitosan, sodium alginate.
The said polyelectrolyte A of step (2) is the PAH hydrochlorate, and said polyelectrolyte B is a kayexalate.
Further; Step (2) is carried out as follows: the albumin calcium carbonate microparticle that step (1) is obtained carries out after with deionized water wash as follows: the deposition (being protic acid calcium microgranule) of 1. getting after the washing is dispersed in 0.5～2mg/mL (preferred 2mg/mL) PAH hydrochlorate aqueous solution; Room temperature (20 ℃) stirs hatches 15min; The centrifugal 5min of 4000r/min gets the deposition washing, the deposition after obtaining to wash; The quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL (preferred 100mg/mL) with the volume of PAH hydrochlorate aqueous solution; 2. the precipitation after will washing again be dispersed in the isopyknic 0.5～2mg/mL of PAH hydrochlorate aqueous solution (preferred 2mg/mL) kayexalate aqueous solution in; Room temperature (20 ℃) stirs and hatches 15min, and the centrifugal 5min of 4000r/min gets the deposition washing; Deposition after water intaking is washed promptly obtains to accomplish the albumin calcium carbonate microparticle of 1 double-layer coatings; 3. will accomplish 1 albumin calcium carbonate microparticle repeatable operation behind the coating 1. with 2. step 3～5 time, accomplish repeatedly double-layer coatings, centrifugal; Get the albumin calcium carbonate microparticle washing behind the final coating, obtain the colloidal particles of nucleocapsid structure.
The said polyelectrolyte A of step (2) is a chitosan, and said polyelectrolyte B is a sodium alginate.
Further; Step (2) is carried out as follows: it is to carry out as follows behind 5.0 the 0.2mol/L NaCl solution washing that the albumin calcium carbonate microparticle that step (1) is obtained uses pH value: 1. get deposition (being the albumin calcium carbonate microparticle) after the washing and be dispersed in the mixed solution that pH value is 5.0 chitosan and 0.5mol/L NaCl aqueous solution; The final concentration of chitosan is 0.5～2mg/mL (preferred 1mg/mL) in the said mixed solution; Room temperature (20 ℃) stirs hatches 15min; The centrifugal 5min of 4000r/min, getting deposition, to use pH value be 5.0 0.2mol/L NaCl solution washing, obtains the deposition after the washing; The quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL (preferred 100mg/mL) with the volume of the mixed solution of chitosan and NaCl aqueous solution; 2. the precipitation after will washing again is dispersed in the mixed solution that pH value is 5.0 sodium alginate and 0.5mol/L NaCl aqueous solution; The final concentration of sodium alginate is 0.5～2mg/mL (preferred 1mg/mL) in the said mixed solution, and room temperature (20 ℃) stirs hatches 15min, the centrifugal 5min of 4000r/min; Getting deposition, to use pH value be behind 5.0 the 0.2mol/L NaCl solution washing; Get the albumin calcium carbonate microparticle that deposition after the washing promptly obtains to accomplish 1 double-layer coatings, the volume of the said sodium alginate and the mixed solution of NaCl aqueous solution is identical with the volume of the mixed solution of chitosan and NaCl aqueous solution, 3. will accomplish 1 albumin calcium carbonate microparticle repeatable operation behind the double-layer coatings 1. with 2. step 3～5 time; Then the albumin calcium carbonate microparticle behind the final coating is added in the glutaraldehyde water solution of volumetric concentration 1%; Stirring at room 12h accomplishes repeatedly double-layer coatings, and is centrifugal; Getting deposition, to use pH value be 5.0 0.2mol/L NaCl solution washing, obtains the colloidal particles of nucleocapsid structure.
The said nucleocapsid structure colloidal particles of step (3) use the EDTA solution pitting method of 0.2mol/L to be: the nucleocapsid structure colloidal particles are dispersed in the EDTA solution of 0.2mol/L; Stir 15～30min; Centrifugal, get the EDTA solution that deposition is scattered in 0.2mol/L and repeat aforesaid operations 3～5 times, calcium carbonate is fully dissolved; Centrifugal, get the precipitate with deionized water washing and promptly accomplish the enucleation process.And then the microcapsule after the enucleation (i.e. deposition) is scattered in heating makes the inner albumin gelation of microcapsule promptly get said pH responsive type polyelectrolyte microcapsule drug administration carrier in the deionized water.
The suitable various pH value scopes of pH responsive type polyelectrolyte microcapsule drug administration carrier according to the invention are generally 2～12.
The drug loading of pH responsive type polyelectrolyte microcapsule drug administration carrier according to the invention is represented with drug level in the microcapsule, calculates with formula (1), and method of testing is:
Said pH responsive type polyelectrolyte microcapsule (like bovine serum albumin white gel microcapsule) is added in the centrifuge tube, and the quantity of gel microcapsule is (2.5 ± 0.3) * 10 in every centrifuge tube
7Individual; Add drug solution (like amycin) again, stirring at room is hatched 12h, and is centrifugal; Get supernatant; The deionized water dilution, ultraviolet spectrophotometer is measured the absorbance of medicine maximum absorption wave strong point (like amycin 253nm place), calculates (like amycin) concentration of supernatant Chinese medicine according to the standard curve of contained medicine (like amycin (DOX)).
C
c=(C
fV
f-C
sV
s)/V
cFormula (1)
C in the formula (1)
c, V
cRepresent the drug level in the microcapsule and the cumulative volume of microcapsule behind the medicine carrying respectively, C
f, V
fRepresent administration concentration and administration volume respectively, C
s, V
sRepresent the volume of supernatant Chinese medicine concentration and supernatant respectively.Under laser confocal microscope, select 100 microcapsules to measure diameter at random, average, and calculate the cumulative volume of microcapsule thus.The quantity of gel microcapsule obtains at microscopically range estimation reading through blood cell calculator, gets meansigma methods three times.
The medicine carrying release performance investigation method of pH responsive type polyelectrolyte microcapsule drug administration carrier according to the invention is: said pH responsive type polyelectrolyte microcapsule (like bovine serum albumin white gel microcapsule) is placed centrifuge tube, and the quantity of gel microcapsule is (2.5 ± 0.3) * 10 in every centrifuge tube
7Individual, add drug solution (like amycin), after stirring at room is hatched 12h, centrifugal; Get precipitate with deionized water washing once, remove supernatant, respectively in the centrifuge tube the different pH value of adding (pH 2.0,5.0; 7.4) the PBS buffer, place 37 ℃ constant-temperature shaking case, hatch and make drug release, at interval certain hour; Gel microcapsule suspension is centrifugal, take out supernatant, add the corresponding equivalent PBS buffer of pH value simultaneously, to keep the delivery systme constancy of volume.With the PBS buffer dilution of supernatant with corresponding pH value; Measure the absorbance that contained medicine maximum absorption wavelength (like amycin 253nm) is located; Standard curve according to contained medicine (like DOX) calculates supernatant Chinese medicine (like amycin) concentration, draws the cumulative release curve of medicine.Get the average of three groups of parallel laboratory test data under each pH condition, draw release profiles.
Room temperature according to the invention is generally 20 ℃.
Compared with prior art; Beneficial effect of the present invention is mainly reflected in: the present invention is pre-filled in the albumin gel in the polyelectrolyte microcapsule; On the one hand because albumin is a kind of polyampholyte, its electrically chargedly change along with the variation of pH; Thereby can control of load and the release of this microcapsule through regulating pH to medicine; When the albumin gel can be during the opposite charge of electrically charged and drug molecule through electrostatic attraction with the ground enrichment of drug molecule high concentration to microcapsule inside, greatly improve the envelop rate and the drug loading of microcapsule, when change pH weakens the electrostatic attraction effect of albumin gel and drug molecule; Medicine can discharge again quickly, thereby reaches intelligent effect; On the other hand, utilize the RF store medication of albumin gel, can reduce prominent the releasing of medicine that cause because the inside and outside drug level gradient of microcapsule is excessive; The inventive method is reasonable in design, and preparation technology is simple, has excellent research and application prospect.
(4) description of drawings
Fig. 1 is the preparation sketch map of pH responsive type polyelectrolyte microcapsule drug administration carrier;
Fig. 2 is laser confocal microscope (CLSM) picture (shown in the A) and embodiment 2 albumin calcium carbonate microparticle scanning electron microscope (SEM) pictures (shown in the B) of the albumin calcium carbonate microparticle of embodiment 3 marked by fluorescein isothiocyanate;
Fig. 3 is transmission electron microscope (TEM) picture (shown in the A) and the SEM picture (shown in the B) of embodiment 4pH responsive type polyelectrolyte microcapsule drug administration carrier;
Fig. 4 is the CLSM picture of the pH responsive type polyelectrolyte microcapsule drug administration carrier of load amycin under embodiment 7 condition of different pH: pH 2.0 (shown in the A); PH 6.5 (shown in the B);
Fig. 5 is the graph of a relation of the interior DOX concentration of pH responsive type polyelectrolyte microcapsule drug administration carrier behind different pH value of embodiment 7 amycin solution and the load DOX, and abscissa is a pH value, and vertical coordinate is a DOX concentration in the microcapsule drug administration carrier,
*There is significant difference in expression P＜0.05;
Fig. 6 is the curve chart of DOX concentration in DOX concentration and the supernatant in the pH responsive type polyelectrolyte microcapsule drug administration carrier after the embodiment 8 variable concentrations amycin loads, and abscissa is an administration DOX concentration, and vertical coordinate is the DOX concentration that records;
Fig. 7 be under embodiment 9 condition of different pH amycin from the releasing curve diagram of pH responsive type polyelectrolyte microcapsule drug administration carrier.
(5) specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the preparation of bovine serum albumin calcium carbonate microparticle
Prepare the calcium chloride water of 0.025mol/L and each 100mL of aqueous sodium carbonate of 0.025mol/L respectively.The bovine serum albumin (BSA) (Nanjing Ao Duofuni bio tech ltd) that takes by weighing 50mg is dissolved in the 0.025mol/L calcium chloride water of 100mL; Under stirring condition, add the 0.025mol/L aqueous sodium carbonate of 100mL rapidly, continue to stir 30s, gained solution left standstill 10min; The centrifugal 5min of 4000r/min; After the precipitate with deionized water washing, vacuum drying 48h promptly gets the about 250mg of bovine serum albumin calcium carbonate microparticle.
Embodiment 2: the preparation of bovine serum albumin calcium carbonate microparticle
Prepare the calcium chloride water of 0.33mol/L and each 100mL of aqueous sodium carbonate of 0.33mol/L respectively.The bovine serum albumin (BSA) (Nanjing Ao Duofuni bio tech ltd) that takes by weighing 500mg is dissolved in the 100mL 0.33mol/L calcium chloride water, under stirring condition, adds the 0.33mol/L aqueous sodium carbonate of 100mL rapidly, continues to stir 30s; Gained solution left standstill 10min, the centrifugal 5min of 4000r/min is after the precipitate with deionized water washing; Vacuum drying 48h; Promptly get the about 3.3g of bovine serum albumin calcium carbonate microparticle, bovine serum albumin calcium carbonate microparticle SEM figure sees shown in the B among Fig. 2.
Embodiment 3: the preparation of the bovine serum albumin calcium carbonate microparticle of Fluorescein isothiocyanate (FITC) labelling
Prepare the calcium chloride water of 0.33mol/L and each 100mL of aqueous sodium carbonate of 0.33mol/L respectively.The bovine serum albumin (BSA) that takes by weighing 500mg Fluorescein isothiocyanate (FITC) labelling is dissolved in the 100mL 0.33mol/L calcium chloride water; Under stirring condition, add the 0.33mol/L aqueous sodium carbonate of 100mL rapidly, continue to stir 30s, gained solution left standstill 10min; The centrifugal 5min of 4000r/min; After the precipitate with deionized water washing, vacuum drying 48h promptly gets the about 3.3g of bovine serum albumin calcium carbonate microparticle of FITC labelling; The CLSM figure of the bovine serum albumin calcium carbonate microparticle of FITC labelling sees shown in the A among Fig. 2.
The preparation of embodiment 4:pH responsive type polyelectrolyte microcapsule drug administration carrier-bovine serum albumin white gel microcapsule
PAH hydrochlorate (PAH) and kayexalate (PSS) are used deionized water dissolving respectively, be mixed with the PAH aqueous solution of 2mg/mL and the PSS aqueous solution of 2mg/mL.Take by weighing the exsiccant bovine serum albumin calcium carbonate microparticle of about 100mg of embodiment 2 preparations, use deionized water wash, the centrifugal 5min of 4000r/min; Deposition repeated washing 3 times is dispersed in the above-mentioned PAH aqueous solution of 1mL room temperature (20 ℃) with the microgranule (deposition) after washed and hatches 15min, then the centrifugal 5min of 4000r/min; Remove supernatant, get behind the deposition reuse deionized water wash centrifugally, repeat 3 times; To wash microgranule (deposition) after 3 times again and be dispersed in the above-mentioned PSS aqueous solution of 1mL room temperature (20 ℃) and hatch 15min, the centrifugal 5min of 4000r/min removes supernatant; It is centrifugal to get precipitate with deionized water washing back, repeats 3 times, gets the albumin calcium carbonate microparticle that deposition promptly obtains to accomplish 1 double-layer coatings; Repeat above-mentioned coating 4 times, promptly get the colloidal particles of nucleocapsid structure.In the colloidal particles of above-mentioned nucleocapsid structure, add 0.2mol/L EDTA aqueous solution; Stir 15min; Centrifugal; Get the EDTA aqueous solution that deposition is scattered in 0.2mol/L once more and repeat aforesaid operations 3 times, get precipitate with deionized water washing 3 times, promptly get the polyelectrolyte microcapsule of filling the bovine serum albumin molecule.At last, the polyelectrolyte microcapsule of filling the bovine serum albumin molecule is scattered in the deionized water 80 ℃ of agitating heating 1h; Make the abundant gelation of albumin molecule; Centrifugal, deposition is the polyelectrolyte microcapsule that is pre-charged with the bovine serum albumin white gel, and we abbreviate " bovine serum albumin white gel microcapsule " as; Be pH responsive type polyelectrolyte microcapsule drug administration carrier, the preparation sketch map is seen shown in Figure 1.
Embodiment 5:pH responsive type polyelectrolyte microcapsule drug administration carrier--the preparation of bovine serum albumin white gel microcapsule
Chitosan (CTS) and sodium alginate (ALG) are dissolved with 0.5mol/L NaCl aqueous solution respectively; Obtain the CTS-NaCl aqueous solution of CTS final concentration 1mg/mL and the ALG-NaCl aqueous solution of ALG final concentration 1mg/mL respectively, and to use 0.1mol/L HCl aqueous solution or 0.1mol/L NaOH aqueous solution to regulate pH value respectively be 5.0.
Take by weighing the exsiccant bovine serum albumin calcium carbonate microparticle of about 100mg of embodiment 2 preparations, with 0.2mol/L NaCl aqueous solution (pH 5.0) washing, 4000r/min is centrifugal, and 5min removes supernatant, repeats 3 times.Bovine serum albumin calcium carbonate microparticle after washed is dispersed in the above-mentioned CTS-NaCl aqueous solution of 1mL room temperature (20 ℃) hatches 15min, the centrifugal 5min of 4000r/min removes supernatant then; Get behind the deposition reuse 0.2mol/LNaCl solution washing centrifugally, repeat 3 times, the microgranule (deposition) after will washing again is dispersed in the above-mentioned ALG-NaCl aqueous solution of 1mL room temperature (20 ℃) and hatches 15min; Centrifugal removal supernatant is got deposition with centrifugal behind the 0.2mol/L NaCl solution washing, repeats 3 times; Get the albumin calcium carbonate microparticle that deposition promptly obtains to accomplish 1 double-layer coatings; Repeat above-mentioned coating 4 times, in the colloidal particles of above-mentioned final coating, add 1mL volumetric concentration 1% glutaraldehyde water solution again, room temperature (20 ℃) stirring reaction 12h makes chitosan crosslinked; Accomplish 4 times coating; 4000r/min is centrifugal, and 5min removes supernatant, and precipitate with deionized water is washed 3 times, promptly gets the colloidal particles of nucleocapsid structure.Then; In the colloidal particles of above-mentioned nucleocapsid structure, add the 0.2mol/LEDTA aqueous solution, stir 30min, centrifugal; Get the EDTA aqueous solution that deposition is scattered in 0.2mol/L once more and repeat aforesaid operations 3 times; The centrifugal supernatant that goes, precipitate with deionized water is washed 3 times, must fill the polyelectrolyte microcapsule of bovine serum albumin.At last, the polyelectrolyte microcapsule of filling bovine serum albumin is scattered in the deionized water, 80 ℃ of agitating heating 1h make the abundant gelation of albumin molecule, obtain bovine serum albumin white gel microcapsule, i.e. pH responsive type polyelectrolyte microcapsule drug administration carrier.
Embodiment 6: the sign of bovine serum albumin calcium carbonate microparticle and pH responsive type polyelectrolyte microcapsule drug administration carrier
(1) the bovine serum albumin calcium carbonate microparticle with embodiment 2 preparations places deionized water to process suspension, suspension is dripped on coverslip natural drying; Metal spraying adopts scanning electron microscope (SEM) (SIRION, FEI; Netherlands) observe, the result sees among Fig. 2 shown in the B; Place deionized water to process suspension the bovine serum albumin calcium carbonate microparticle with Fluorescein isothiocyanate (FITC) labelling of embodiment 3 preparations; Suspension is dropped on the microscope slide; Use the coverslip mounting immediately, at laser confocal microscope (LSM 510 META, ZEISS; Germany) observe down, the result sees among Fig. 2 shown in the A;
(2) the bovine serum albumin white gel microcapsule (pH responsive type polyelectrolyte microcapsule drug administration carrier) with embodiment 4 preparations mixes with deionized water; Processing suspension also drips on the copper mesh that contains carbon film; Natural drying, (JEM 1230, JEOL at transmission electron microscope (TEM); Japan) observe down its form, the result sees among Fig. 3 shown in the A; The bovine serum albumin white gel microcapsule aqueous suspensions of embodiment 4 preparations is dripped on coverslip, natural drying, metal spraying, (SIRION, FEI Netherlands) observe, and the result sees B shown in Fig. 3 to adopt scanning electron microscope (SEM).
The medicine carrying behavior of embodiment 7 bovine serum albumin white gel microcapsules under condition of different pH investigated
Precision takes by weighing an amount of amycin (DOX), and the PBS buffer with different pH value (2.0,4.0,5.0,6.5) is a solvent respectively, and ultrasonic dissolution is made into the DOX solution (pH value is respectively 2.0,4.0,5.0,6.5) that concentration is 1000 μ g/mL.
The quantity of gel microcapsule is (2.5 ± 0.3) * 10 in the equivalent bovine serum albumin white gel microcapsule that adds embodiment 4 preparations respectively in 4 centrifuge tubes, every centrifuge tube
7Individual, (pH value is respectively 2.0,4.0 toward the DOX solution that wherein adds the above-mentioned different pH value that prepare of 500 μ L, 1000 μ g/mL respectively again; 5.0,6.5), room temperature (20 ℃) stirs hatches 12h; Centrifugal; Get 200 μ L supernatants, deionized water is diluted to 10mL, measures the absorbance at 253nm place with ultraviolet spectrophotometer.Average is got in experiment repetition under every group of pH condition 3 times, calculates doxorubicin concentration in the supernatant according to the standard curve of DOX.
The standard curve equation of DOX adopts the absorbance at 253nm place to obtain equally: prepare 5 μ g/mL respectively; 7 μ g/mL, 10 μ g/mL, 12 μ g/mL; The DOX aqueous solution of 17 μ g/mL series concentration; Ultraviolet spectrophotometer is measured the absorbance at 253nm place, with absorbance DOX concentration is carried out linear regression, gets the standard curve equation to be: y=0.0402x+0.023 (r=0.9998).
The drug loading of bovine serum albumin white gel microcapsule is represented with doxorubicin concentration in the microcapsule, asks calculation with formula (1) meter:
C
c=(C
fV
f-C
sV
s)/V
cFormula (1)
C in the formula (1)
c, V
cRepresent the DOX concentration in the microcapsule and the cumulative volume of microcapsule behind the medicine carrying respectively, C
f, V
fRepresent administration concentration and administration volume respectively, C
s, V
sRepresent the DOX concentration of supernatant and the volume of supernatant respectively.Under laser confocal microscope, select 100 microcapsules to measure diameter at random, average, and calculate the cumulative volume of microcapsule thus.The quantity of gel microcapsule obtains at microscopically range estimation reading through blood cell calculator, gets meansigma methods three times.
The result shows that bovine serum albumin white gel microcapsule increases and significantly strengthens with the DOX pH value of solution the load capacity of DOX, at pH 2.0,5.0, all has significant difference under 6.5 conditions, and the result sees Fig. 5.
In addition; PH exists appreciable impact also to carry out checking intuitively through CLSM to the medicine carrying behavior of bovine serum albumin white gel microcapsule: pH 2.0 and pH 6.5 conditions are downloaded into microcapsule behind the DOX carry out CLSM and observe; DOX mainly is adsorbed on the microcapsule surface under pH 2.0 conditions; The red fluorescence of DOX is not seen in microcapsule inside, sees among Fig. 4 shown in the A; And DOX can be deposited on microcapsule inside in a large number under pH 6.5 conditions, sees among Fig. 4 shown in the B.This mainly is because of the DOX positively charged, and BSA positively charged when pH 2.0 is electronegative when pH 6.5, and the electrostatic interaction between BSA and the DOX is the main drive of DOX load.
Embodiment 8 bovine serum albumin white gel microcapsules are investigated the medicine carrying behavior of variable concentrations DOX solution
According to embodiment 7 experimental results, we select pH 6.5 to be loading condition.
Precision takes by weighing an amount of DOX, and the PBS buffer of pH 6.5 is a solvent, and ultrasonic dissolution is made into 50 μ g/mL; 100 μ g/mL, 250 μ g/mL, 500 μ g/mL; The DOX solution of 1000 μ g/mL series concentration, other operations and condition are investigated the load behavior under the different DOX concentration conditions with embodiment 7.
The result shows along with DOX concentration in the increase microcapsule of DOX solution concentration constantly increases, and under all concentration conditions in the microcapsule DOX concentration always than supernatant in the concentration of DOX high tens of times to Radix Achyranthis Bidentatae, the result sees Fig. 6, has shown tangible auto-deposition effect.
The release performance of the bovine serum albumin white gel microcapsule of 9 years DOX of embodiment under condition of different pH investigated
In 3 centrifuge tubes, add the equivalent bovine serum albumin white gel microcapsule that embodiment 4 methods prepare respectively, gel microcapsule quantity is (2.5 ± 0.3) * 10
7Individual, add the DOX solution that 0.5mL concentration is 1000 μ g/mL (pH value is 6.5, and compound method is with embodiment 7) in 3 centrifuge tubes respectively; After room temperature (20 ℃) stirs and hatches 12h, centrifugal, get the precipitate with deionized water washing once; Remove supernatant, in 3 centrifuge tubes, add 1mL PBS buffer (pH=7.4,5.0 respectively; 2.0), placing 37 ℃ constant-temperature shaking case, vibration is hatched and is made drug release; Different time points (15min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h) is centrifugal with gel microcapsule suspension at interval, takes out 800 μ L supernatants; The PBS buffer that adds 37 ℃ of 800 corresponding μ L of pH value simultaneously is to keep the delivery systme constancy of volume.800 μ L supernatants are diluted to 3mL with the PBS buffer of corresponding pH value, are determined at the absorbance at 253nm place, draw the cumulative release curve of amycin.Get the average of three groups of parallel laboratory test data under each pH condition, the result sees Fig. 7.
Fig. 7 result shows that along with the reduction of release medium pH value, the rate of release of DOX enlarges markedly.This mainly is to cause the positive charge of BSA to gradually reduce because pH reduces, and makes that the electrostatic attraction effect between BSA and the DOX reduces gradually.
Claims (9)
1. the method for preparing of a pH responsive type polyelectrolyte microcapsule drug administration carrier is characterized in that said method is: (1) adds albumin that to make albuminous concentration in 0.025mol/L～0.33mol/L calcium chloride water be 0.5mg/mL～5mg/mL, stirring and dissolving; Add and the isopyknic aqueous sodium carbonate of described calcium chloride water same molar ratio; Continue to stir, leave standstill, get deposition; Drying obtains the albumin calcium carbonate microparticle; (2) getting albumin calcium carbonate microparticle that step (1) obtains operates as follows and 1. is dispersed in 0.5mg/mL～2mg/mL polyelectrolyte A solution; Stirring at room is hatched 10～20min; Centrifugalize; Get washing of precipitate, the quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL with the volume of polyelectrolyte A solution; 2. the precipitation after will washing again is dispersed in 0.5mg/mL～2mg/mL and the isopyknic polyelectrolyte B of the polyelectrolyte A solution solution, and stirring at room is hatched 10～20min, centrifugalize; Get washing of precipitate, get the albumin calcium carbonate microparticle that deposition after the washing promptly obtains accomplishing 1 double-layer coatings, 3. with the albumin calcium carbonate microparticle repeatable operation behind 1 coating 1. with 2. step 3～5 time; Accomplish repeatedly double-layer coatings; Centrifugal, washing, the colloidal particles of acquisition nucleocapsid structure; Said polyelectrolyte A and polyelectrolyte B are two kinds of polyelectrolyte with opposite charges; The colloidal particles of the nucleocapsid structure that (3) step (2) is obtained dissolve enucleation with the EDTA solution of 0.2mol/L; Centrifugal, to get deposition and be scattered in the deionized water, 70～80 ℃ of heating make the abundant gelation of albumin molecule; Centrifugal, deposition is said pH responsive type polyelectrolyte microcapsule drug administration carrier.
2. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1 is characterized in that the said albumin of step (1) is bovine serum albumin or human serum albumin.
3. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1 is characterized in that the concentration of the said albumin of step (1) in 0.025mol/L～0.33mol/L calcium chloride water is 2.0mg/mL～3.0mg/mL.
4. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1; It is characterized in that step (1) gets intermediate processing and be: reaction with the centrifugal 5～10min of reactant liquor 4000～7000r/min, is got deposition after finishing; Vacuum drying 48～72h obtains the albumin calcium carbonate microparticle.
5. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1 is characterized in that the said polyelectrolyte A of step (2) is the PAH hydrochlorate, and said polyelectrolyte B is a kayexalate.
6. like the method for preparing of claim 1 or 5 said pH responsive type polyelectrolyte microcapsule drug administration carriers; It is characterized in that step (2) carries out as follows: the albumin calcium carbonate microparticle that step (1) is obtained carries out after with deionized water wash as follows: the albumin calcium carbonate microparticle of 1. getting after the washing is dispersed in the 2mg/mL PAH hydrochlorate aqueous solution; Stirring at room is hatched 15min; The centrifugal 5min of 4000r/min gets the deposition washing, the deposition after obtaining to wash; The quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL with the volume of PAH hydrochlorate aqueous solution; 2. the precipitation after will washing again be dispersed in the isopyknic 2mg/mL kayexalate of PAH hydrochlorate aqueous solution aqueous solution in; Stirring at room is hatched 15min, and the centrifugal 5min of 4000r/min gets the deposition washing; Deposition after water intaking is washed promptly obtains to accomplish the albumin calcium carbonate microparticle of 1 double-layer coatings; 3. with the albumin calcium carbonate microparticle repeatable operation behind 1 coating 1. with 2. step 3～5 time, accomplish repeatedly double-layer coatings, centrifugal; Get the albumin calcium carbonate microparticle washing behind the final coating, obtain the colloidal particles of nucleocapsid structure.
7. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1 is characterized in that the said polyelectrolyte A of step (2) is a chitosan, and said polyelectrolyte B is a sodium alginate.
8. like the method for preparing of claim 1 or 7 said pH responsive type polyelectrolyte microcapsule drug administration carriers; It is characterized in that step (2) carries out as follows: it is to carry out as follows behind 5.0 the 0.2mol/L NaCl solution washing that the albumin calcium carbonate microparticle that step (1) is obtained uses pH value: 1. get albumin calcium carbonate microparticle after the washing and be dispersed in the mixed solution that pH value is 5.0 chitosan and 0.5mol/L NaCl aqueous solution; The final concentration of chitosan is 1mg/mL in the said mixed solution; Stirring at room is hatched 15min; The centrifugal 5min of 4000r/min, getting deposition, to use pH value be 5.0 0.2mol/L NaCl solution washing, obtains the deposition after the washing; The quality consumption of said albumin calcium carbonate microparticle is counted 100～200mg/mL with the volume of the mixed solution of chitosan and NaCl aqueous solution; 2. the precipitation after will washing again is dispersed in the mixed solution that pH value is 5.0 sodium alginate and 0.5mol/L NaCl aqueous solution; The final concentration of sodium alginate is 1mg/mL in the said mixed solution, and stirring at room is hatched 15min, the centrifugal 5min of 4000r/min; Getting deposition, to use pH value be behind 5.0 the 0.2mol/L NaCl solution washing; Get the albumin calcium carbonate microparticle that deposition promptly obtains to accomplish 1 double-layer coatings, the volume of the said sodium alginate and the mixed solution of NaCl aqueous solution is identical with the volume of the mixed solution of chitosan and NaCl aqueous solution, 3. will accomplish 1 albumin calcium carbonate microparticle repeatable operation behind the double-layer coatings 1. with 2. step 3～5 time; Then the deposition behind the final coating is added in the glutaraldehyde water solution of volumetric concentration 1%; Stirring at room 12h accomplishes repeatedly double-layer coatings, and is centrifugal; Getting deposition, to use pH value be 5.0 0.2mol/L NaCl solution washing, obtains the colloidal particles of nucleocapsid structure.
9. the method for preparing of pH responsive type polyelectrolyte microcapsule drug administration carrier according to claim 1; It is characterized in that the said nucleocapsid structure colloidal particles of step (3) use the EDTA solution pitting method of 0.2mol/L to be: the nucleocapsid structure colloidal particles are dispersed in the EDTA solution of 0.2mol/L, stir 15～30min, centrifugal; Get the EDTA solution that deposition is scattered in 0.2mol/L and repeat aforesaid operations 3～5 times; Calcium carbonate is fully dissolved, centrifugal, get the precipitate with deionized water washing and promptly accomplish the enucleation process.
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