CN105664242A - Method for preparing PLGA microspheres with porous surfaces - Google Patents

Abstract

The invention provides a method for preparing PLGA microspheres with the porous surfaces. The method includes the steps that PLGA is added into an organic solvent, stirred and dissolved, a hole forming solution is dropwise added into the mixture, the mixture is subjected to ultrasonic emulsification, and emulsified liquid is formed, wherein the molar ratio of LA to GA of the PLGA ranges from 50:50 to 90:10; the emulsified liquid is dropwise added into a stirring external water phase, preset-volume deionized water is added, and continuous stirring is conducted at changed speeds till the organic solvent is completely volatilized, wherein the external water phase is formed in the mode that a water-soluble surfactant is added into deionized water; the solution obtained after the organic solvent is completely volatilized is centrifuged, and washed with the deionized water, supernatant liquid is removed, and the PLGA microspheres are obtained; a NaOH solution is added into the PLGA microspheres, and the mixture is mixed to be even and placed to a shaking table to continue to be reacted; the reacted PLGA microspheres are centrifuged and washed with deionized water repeatedly, and frozen and dried, and the PLGA microspheres with the porous surfaces are obtained. The method is simple in technology, short in period and suitable for industrialized mass production.

Description

The PLGA method for preparing microsphere of porous surface

Technical field

The present invention relates to porous polymer bead field of material technology, more specifically, relate to a kind of W/O/W of utilization double emulsification solvent volatility process and prepare, in conjunction with directionally hydrolyzing technology, the method that surface has the injectable cell microsphere carrier of macroporous structure.

Background technology

Poly lactic-co-glycolic acid (PLGA) is that a kind of degradable biological high molecular polymer, its biomechanics and degradability can pass through the monomer lactic acid (LA) of its polymerization and the ratio of hydroxyacetic acid and Molecular weight regulation. Due to good biocompatibility, it is approved by the FDA in the united states and can be used for clinic, have a wide range of applications in biomedical engineering field, be made at present as the absorbable bone screw of different kind organism, operation suture thread, pharmaceutical carrier and tissue engineering bracket material. It is made into the microsphere that form, surface porosity are controlled, by being a kind of good injectable organizational project cell carrier, there is potential application widely.

Microsphere is the novel cell carrier that development in recent years is got up, and it is the spheroid carrier made with biomaterials such as albumen, gelatin, polylactic acid. Micro-sphere material is that the development of current technical field of biological material is very fast and one of material that application prospect is extraordinary, compared with conventional biomaterial, microsphere has following features and performance: 1) make larger-diameter duct in microsphere, so as to cells survival can be held, microsphere is a kind of tissue engineering bracket, is applied to minimally-invasive treatment as injectable timbering material; 2) being modified by microsphere surface, the factors such as ambient temperature, pH, magnetic field, ultrasonic, radiation can be produced quickly reaction by microsphere; 3) microsphere as porogen, can improve the porosity of conventional blocks timbering material, is conducive to growing into of biological tissue, generates the carrying out of metabolism and improves the mechanical property of material; 4) microsphere is as microreactor, and induction apatite, in its surface mineralising, is conducive to osteanagenesis, is good bone renovating material; 5) microsphere spherical attribute inherently makes it have good syringeability, can be used for minimally-invasive treatment the easy molding when repairing the Cranial defect of complex region;6) there is micro-/ nano size. Owing to having less size and bigger specific surface area, it is widely used as medicine controlled release carrier; 7) microsphere can cover adverse drug abnormal smells from the patient and taste, improves the stability of medicine, it is prevented that medicine is at gastric inactivation or reduces the medicine stimulation to stomach, slow release or controlled release and targeting.

It is currently used for the physico-chemical process preparing microsphere by there being 3 big classes: phase separation method, miniflow Dow process and solvent evaporated method. The method being separated to use substantial amounts of organic solvent as flocculating agent, and the removal of these organic solvents is comparatively difficult, can bring environmental pollution and cytotoxicity, and the cell carrier higher as biocompatibility requirement is very not applicable. Miniflow Dow process needs complex fluid system, requires higher to preparation condition. And solvent evaporated method is a kind of comparatively conventional method for preparing microsphere, application is comparatively general at present, the method is to remove organic solvent from emulsion and make polymeric hardener balling-up, microspherulite diameter can be controlled preferably, by addition in emulsion process, there is chemosmotic material and can control its surface porosity as porogen. The method does not need special high tension unit without drawing the poisonous flocculating agent that is separated. But microsphere prepared by the method, porous surface aperture is micron order mostly, it is more difficult to generate the about 20 μm of macroporous structures being available for cell entrance that aperture is controlled. It has developed a kind of new method in above-mentioned analysis.

Summary of the invention

In view of the above problems, it is an object of the invention to provide the PLGA method for preparing microsphere of a kind of porous surface, technique is simple, and the cycle is short, is suitable for using industrial large-scale production.

The present invention provides the PLGA method for preparing microsphere of a kind of porous surface, including: PLGA is joined in organic solvent, after stirring and dissolving, be added dropwise to pore solution ultrasonic emulsification, form emulsion; Wherein, the molar ratio of LA and the GA of described PLGA is: 50:50～90:10;

Being added dropwise to by described emulsion in the outer aqueous phase of stirring, and add the deionized water of preset vol, change speed continuously stirred, until described organic solvent volatilizees completely, wherein, described outer aqueous phase adds in deionized water for water soluble surfactant active;

Solution after being volatilized completely by described organic solvent is centrifuged, and cleans with deionized water, removes supernatant, it is thus achieved that PLGA microsphere;

NaOH solution is joined in described PLGA microsphere, mix homogeneously, and it is put into shaking table continuation reaction;

By reacted PLGA microsphere deionized water centrifuge washing repeatedly, lyophilization, it is thus achieved that the PLGA microsphere of porous surface.

Furthermore it is preferred that scheme be that the molecular weight of described PLGA is 10000 to 100000.

Furthermore it is preferred that scheme be that described pore solution is bovine serum albumin or PVA aqueous solution, and the concentration of described pore solution is 0.1-4%.

Furthermore it is preferred that scheme be that described organic solvent is dichloromethane, chloroform, acetonitrile, ethyl acetate, the concentration of described organic solvent is 0.25-10%.

Furthermore it is preferred that scheme be that described water soluble surfactant active is PVA, carboxymethyl cellulose or PEG; The concentration of described water soluble surfactant active is 0.1-2%.

Furthermore it is preferred that scheme be that, when forming emulsion, oil-water ratio is 1:3～1:5, and quickly stir after formation colostrum through 10-50min, add the deionized water of 10 times and change speed and continue stir about 4h, to the volatilization of described organic solvent completely.

Furthermore it is preferred that scheme be, in NaOH solution is joined described PLGA microsphere, mix homogeneously, and be put into shaking table continue reaction process in,

The NaOH solution that concentration is 0.1～1Mol/L is joined in described PLGA microsphere, at 5～40 DEG C, make described PLGA microsphere surface aperture generation hydrolysis, form macropore.

From technical scheme above it can be seen that the PLGA method for preparing microsphere of porous surface provided by the invention, the PLGA microsphere of the porous surface of formation is that surface has the macroporous structure being available for cell entrance, the PLGA porous microsphere that aperture is about 20 μm, uniform particle sizes, internal void is through, and appearance structure is unified; Adopt when emulsifying the method for ultrasonic emulsification to shorten emulsification times, adopt the albumin and PVA aqueous solution that biocompatibility is good as porogen, acellular toxic and side effects; Later stage is in conjunction with NaOH alkaline solution directionally hydrolyzing, the pore size controlling surface is 20 μm, the shortcoming that the microsphere aperture only prepared is less than normal can be improved by emulsion process, prepared microsphere macropore diameter meets the requirement that cell enters, microsphere good sphericity, particle diameter about 100 μm, has good syringeability.

In order to realize above-mentioned and relevant purpose, one or more aspects of the present invention include the feature that will be explained in below and be particularly pointed out in the claims. Description below and accompanying drawing describe some illustrative aspects of the present invention in detail. But, some modes in the various modes that only can use principles of the invention of these aspects instruction. Additionally, it is contemplated that include all these aspects and their equivalent.

Accompanying drawing explanation

By the content of the reference explanation below in conjunction with accompanying drawing and claims, and along with being more fully understood from the present invention, other purpose of the present invention and result will be more apparent and should be readily appreciated that. In the accompanying drawings:

Fig. 1 is the PLGA method for preparing microsphere schematic flow sheet of porous surface according to embodiments of the present invention;

Fig. 2 is the scanning electron microscopic picture of the PLGA microsphere of porous surface according to embodiments of the present invention.

Label identical in all of the figs indicates similar or corresponding feature or function.

Detailed description of the invention

In the following description, for purposes of illustration, in order to provide the comprehensive understanding to one or more embodiments, many details are elaborated. It may be evident, however, that these embodiments can also be realized when not having these details.

Below with reference to accompanying drawing, specific embodiments of the invention are described in detail.

In order to the PLGA method for preparing microsphere of porous surface provided by the invention is described, Fig. 1 illustrates the PLGA method for preparing microsphere flow process of porous surface according to embodiments of the present invention.

As it is shown in figure 1, the PLGA method for preparing microsphere of porous surface provided by the invention includes:

S110: joined by PLGA in organic solvent, after stirring and dissolving, is added dropwise to pore solution ultrasonic emulsification, forms emulsion; Wherein, the molar ratio of LA and the GA of described PLGA is: 50:50～90:10;

S120: emulsion is added dropwise in the outer aqueous phase of stirring, and add the deionized water of preset vol, change speed continuously stirred, until organic solvent volatilizees completely, wherein, outer aqueous phase adds in deionized water for water soluble surfactant active;

S130: the solution after being volatilized completely by organic solvent is centrifuged, and clean with deionized water, remove supernatant, it is thus achieved that PLGA microsphere;

S140: NaOH solution is joined in described PLGA microsphere, mix homogeneously, and it is put into shaking table continuation reaction;

S150: by reacted PLGA microsphere deionized water centrifuge washing repeatedly, lyophilization, it is thus achieved that the PLGA microsphere of porous surface.

Said method is method detailed prepared by the PLGA microsphere of porous surface, and wherein, the molecular weight of PLGA is 10000 to 100000. Porogen is bovine serum albumin or PVA aqueous solution, it is preferable that PVA, and porogen concentration is 0.1-4% (w/v); Organic solvent is dichloromethane, chloroform, acetonitrile, ethyl acetate, it is preferable that dichloromethane; The concentration of PLGA organic solvent is 0.25-10% (w/v).

It addition, it is PVA that property agent is closed on aqueous phase surface, carboxymethyl cellulose or PEG, it is preferable that PVA, PVA molecular weight is 40000-100000, and its concentration controls at 0.1-2% (w/v).

In step s 110, using obtained porogen aqueous solution as interior aqueous phase, join under ultrasound condition in oil phase, form water-in-oil emulsion (during water-in-oil emulsion, have employed ultrasonic emulsification quickly to form emulsion), ultrasonic power is 8-40W, and the time controls at 30s-5min. Aqueous phase solution in porogen: oil-phase solution=1:10～20 (v/v).

In the step s 120, when forming emulsion, oil-water ratio is 1:3～1:5, and after 10-50min quickly forms colostrum with the stirring of 800-1600rpm speed, the deionized water adding 10 times continues with 200-500rpm speed stir about 2-4h, to the volatilization of described organic solvent completely.

In step S140, the NaOH solution that concentration is 0.1～1Mol/L is joined in described PLGA microsphere, at 5～40 DEG C, make described PLGA microsphere surface aperture generation hydrolysis, form macropore; The ratio of the aqueous slkali and microsphere that process microsphere controls at concentration 1-5% (w/v), and the response time is 2min-30min.

Fig. 2 illustrates the scanning electron microscopic picture of the PLGA microsphere of porous surface according to embodiments of the present invention, and this picture is the electron microscopic picture of the PLGA microsphere of the porous surface that the flow process according to above-mentioned Fig. 1 is prepared.

The method flow adopting Fig. 1 is prepared the PLGA microsphere of porous surface by following example, and wherein, the raw material sources that embodiment adopts illustrate, PLGA is purchased from Jinan, Shandong Province Dai Gang bio tech ltd; PVA is purchased from Sigma-Aldrich; Dichloromethane, chloroform, ethyl acetate, acetonitrile, carboxymethyl cellulose, Polyethylene Glycol is purchased from Chengdu Ke Long chemical reagent factory; Ultrasonic emulsification machine used is purchased from the raw analysis in Shanghai.

Example one

The present embodiment takes following steps:

1) by 100mgPLGA (50:50), join in 10mL dichloromethane, be stirred continuously and be completely dissolved to it;

2) prepare 1wt% PVA solution (Mw=80000), with liquid-transfering gun draw 1mLPVA solution, when ultrasound intensity is 24W, be added dropwise to step 1) in oil phase in form water in oil emulsion, ultrasonic time 60s;

3) by step 2) in gained emulsion stirring when join in 30mLPVA aqueous solution, stir speed (S.S.) is 800rpm, stirring 30min after, add 300mL deionized water wherein, change stir speed (S.S.) is 400rpm, continues stirring 4h, volatilizees completely to dichloromethane.

4) centrifugal collection PLGA microsphere, and wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min.

5) in step 4) to join with microspheres quality volume ratio in the PLGA microsphere of gained be 1%, concentration is the NaOH of 0.2M, stirs, and puts into and continues reaction 5min on shaking table, and controlling reaction temperature is 30 DEG C;

6) recentrifuge wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min. Collect microsphere lyophilization, there is 15 ± 10 μm of macroporous structures and injectable porous microsphere cell carrier that particle diameter is 60 ± 20 μm to surface.

Example two

The present embodiment takes following steps:

1) by 80mgPLGA (65:35), join in 12mL chloroform, be stirred continuously and be completely dissolved to it;

2) prepare 1wt% BSA solution, with liquid-transfering gun draw 1mLBSA solution, when ultrasound intensity is 32W, be added dropwise to step 1) in oil phase in form water in oil emulsion, ultrasonic time 50s;

3) by step 2) in gained emulsion stirring when join in 30mLPVA1wt% aqueous solution, stir speed (S.S.) is 800rpm, stirring 30min after, add 300mL deionized water wherein, change stir speed (S.S.) is 400rpm, continues stirring 4h, volatilizees completely to chloroform.

4) centrifugal collection PLGA microsphere, and wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min.

5) in step 4) to add with microspheres quality volume ratio in the PLGA microsphere of gained be 1%, concentration is the NaOH of 0.2M, stirs, and puts into and continues reaction 5min on shaking table, and controlling reaction temperature is 35 DEG C;

6) recentrifuge is washed 3 times, and rotating speed is 4500rpm, and the time is 10min. Collect microsphere lyophilization, there is 20 μm of macroporous structures and the injectable porous microsphere cell carrier of particle diameter about 100 μm to surface.

Example three

The present embodiment takes following steps:

1) by 50mgPLGA (75:25), join in 5mL ethyl acetate, be stirred continuously and be completely dissolved to it;

2) prepare 1wt% PVA solution, with liquid-transfering gun draw 0.5mLPVA solution, when ultrasound intensity is 24W, be added dropwise to step 1) in oil phase in form water in oil emulsion, ultrasonic time 60s;

3) by step 2) in gained emulsion stirring when join in 15mL0.1wt% carboxymethyl cellulose aqueous solution, stir speed (S.S.) is 800rpm, after stirring 30min, add 150mL deionized water wherein, change stir speed (S.S.) to 400rpm, continue stirring 4h, volatilize completely to ethyl acetate.

4) centrifugal collection PLGA microsphere, and wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min.

5) in step 4) to join with microspheres quality volume ratio in the PLGA microsphere of gained be 1%, concentration is the NaOH of 0.1M, stirs, and puts into and continues reaction 30min on shaking table, and controlling reaction temperature is 25 DEG C;

6) recentrifuge is washed 3 times, and rotating speed is 4500rpm, and the time is 10min. Collect microsphere lyophilization, there is 20 μm of macroporous structures and the injectable porous microsphere cell carrier of particle diameter about 100 μm to surface.

Example four

The present embodiment takes following steps:

1) by 70mgPLGA (85:15), join 10mL acetonitrile, be stirred continuously and be completely dissolved to it;

2) prepare 1wt% PVA solution, with liquid-transfering gun draw 1mLPVA solution, when ultrasound intensity is 24W, be added dropwise to step 1) in oil phase in form water in oil emulsion, ultrasonic time 60s;

3) by step 2) in gained emulsion stirring when join in 30mL1wt%PVA aqueous solution, stir speed (S.S.) is 800rpm, stirring 30min after, add 300mL deionized water wherein, change stir speed (S.S.) to 300rpm, continue stirring 4h, volatilize completely to acetonitrile.

4) centrifugal collection PLGA microsphere, and wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min.

5) in step 4) to join with microspheres quality volume ratio in the PLGA microsphere of gained be 1%, concentration is the NaOH of 0.3M, stirs, and puts into and continues reaction 5min on shaking table, and controlling reaction temperature is 20 DEG C;

6) recentrifuge is washed 3 times, and rotating speed is 4500rpm, and the time is 10min. Collect microsphere lyophilization, there is 20 μm of macroporous structures and the injectable porous microsphere cell carrier of particle diameter about 100 μm to surface.

Example five

The present embodiment takes following steps:

1) by 80mgPLGA (90:10), join 10mL dichloromethane, be stirred continuously and be completely dissolved to it;

2) prepare 1wt% PVA solution, with liquid-transfering gun draw 1mLPVA solution, when ultrasound intensity is 25W, be added dropwise to step 1) in oil phase in form water in oil emulsion, ultrasonic time 90s;

3) by step 2) in gained emulsion stirring when join 30mL1wt% Polyethylene Glycol (M_w=2000) in aqueous solution, stir speed (S.S.) is 800rpm, after stirring 30min, adds 300mL deionized water wherein, changes stir speed (S.S.) to 300rpm, continues stirring 4h, volatilize completely to dichloromethane.

4) centrifugal collection PLGA microsphere, and wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min.

5) in step 4) to join with microspheres quality volume ratio in the PLGA microsphere of gained be 1%, concentration is the NaOH of 0.3M, stirs, and puts into and continues reaction 15min on shaking table, and controlling reaction temperature is 15 DEG C;

6) recentrifuge wash 3 times with deionized water, rotating speed is 4500rpm, and the time is 10min. Collect microsphere lyophilization, there is 20 μm of macroporous structures and the injectable porous microsphere cell carrier of particle diameter about 120 μm to surface.

Describe the surface proposed according to the present invention above with reference to accompanying drawing in an illustrative manner and have eurypyloue PLGA micro-sphere material. It will be understood by those skilled in the art, however, that for the proposed surface macropore PLGA microsphere of the invention described above, it is also possible on without departing from the basis of present invention, make various improvement. Therefore, protection scope of the present invention should be determined by the content of appending claims. .

Claims (7)

1. a PLGA method for preparing microsphere for porous surface, including:

PLGA is joined in organic solvent, after stirring and dissolving, be added dropwise to pore solution ultrasonic emulsification, form emulsion; Wherein, the molar ratio of LA and the GA of described PLGA is: 50:50～90:10;

Being added dropwise to by described emulsion in the outer aqueous phase of stirring, and add the deionized water of preset vol, change speed continuously stirred, until described organic solvent volatilizees completely, wherein, described outer aqueous phase adds in deionized water for water soluble surfactant active;

Solution after being volatilized completely by described organic solvent is centrifuged, and cleans with deionized water, removes supernatant, it is thus achieved that PLGA microsphere;

NaOH solution is joined in described PLGA microsphere, mix homogeneously, and it is put into shaking table continuation reaction;

By reacted PLGA microsphere deionized water centrifuge washing repeatedly, lyophilization, it is thus achieved that the PLGA microsphere of porous surface.

2. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, wherein,

The molecular weight of described PLGA is 10000 to 100000.

3. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, wherein,

Described pore solution is bovine serum albumin or PVA aqueous solution, and the concentration of described pore solution is 0.1-4%.

4. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, wherein,

Described organic solvent is dichloromethane, chloroform, acetonitrile, ethyl acetate, and the concentration of described organic solvent is 0.25-10%.

5. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, its China and foreign countries' aqueous phase,

Described water soluble surfactant active is PVA, carboxymethyl cellulose or Polyethylene Glycol;

The concentration of described water soluble surfactant active is 0.1-2%.

6. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, wherein,

In the outer aqueous phase that described emulsion is added dropwise to stirring, and add the deionized water of preset vol, continuously stirred, until during described organic solvent volatilizees completely,

When forming emulsion, oil-water ratio is 1:3～1:5, and quickly stirs after formation colostrum through 10-50min, adds the deionized water of 10 times, changes speed and continues stir about 4h, to the volatilization of described organic solvent completely.

7. the PLGA method for preparing microsphere of porous surface as claimed in claim 1, wherein,

In NaOH solution is joined described PLGA microsphere, mix homogeneously, and it is put in the process that shaking table continues reaction,

The NaOH solution that concentration is 0.1～1Mol/L is joined in described PLGA microsphere, at 5～40 DEG C, make described PLGA microsphere surface aperture generation hydrolysis, form macropore.