CN111875817A

CN111875817A - Preparation method and application of hollow microspheres

Info

Publication number: CN111875817A
Application number: CN202010831449.8A
Authority: CN
Inventors: 孙振华; 黄珂; 李英文; 施坤宁; 杨慧莹; 蒋雪峰
Original assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Current assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Priority date: 2020-03-20
Filing date: 2020-08-18
Publication date: 2020-11-03

Abstract

The invention belongs to the technical field of high polymer materials and biomedical engineering, and relates to a preparation method and application of hollow microspheres. The invention adopts alcohol (especially ethanol) which is mutually soluble with organic solvent and water as a pore-foaming agent, and prepares the polylactic acid-glycolic acid microspheres by a one-step emulsification method. The microspheres prepared by the method have the characteristics of hollow interior, small density, diameter of 100-500 mu m and narrow diameter distribution; the microspheres with different internal structures can be prepared by adjusting the dosage of the alcohol, and after the surface of the microspheres is modified with a material (such as gelatin) with biological activity, the microspheres have great application value in the aspects of tissue engineering and cell carriers. Alcohol as a pore-foaming agent can form a stable solution with an oil phase without layering to obtain hollow uniform microspheres; meanwhile, the obtained microspheres are small in density and 100-500 mu m in diameter, meet the requirements of three-dimensional cell culture microcarriers and are suitable for cell culture.

Description

Preparation method and application of hollow microspheres

Technical Field

The invention belongs to the technical field of high polymer materials and biomedical engineering, and relates to a preparation method of hollow microspheres, the hollow microspheres prepared by the method, and application of the hollow microspheres.

Background

polylactic-CO-glycolic acid (PLGA) is a biodegradable, safe and nontoxic polymer material formed by random copolymerization of two monomers, namely Lactic Acid (LA) and Glycolic Acid (GA), and is decomposed into CO after being degraded in vivo₂And H₂And O. PLGA has good biocompatibility, is approved by the United states FDA in 1997 to be used as a pharmaceutical adjuvant, and has wide application in the field of tissue engineering.

Because the solid PLGA microspheres have long degradation time in vivo and in vitro, the application range of the solid PLGA microspheres is limited, the preparation of porous microspheres and the acceleration of the degradation of the microspheres become hot spots of research in recent years. The porogens reported to be used for preparing PLGA porous microspheres at present are various, such as sodium bicarbonate (NaHCO)₃) Ammonium hydrogen carbonate (NH)₄HCO₃) Phosphate Buffered Saline (PBS), polyvinyl alcohol (PVA), Pluronic (Pluronic) F-127, gelatin (gelatin), and the like. Chung H.J. et al prepared porous PLGA microspheres using gelatin as a pore-forming agent in a microfluidic manner and used for culturing C2C 12-myoblasts^[1]. KimH.K. et al prepared PLGA microspheres loaded with recombinant human growth hormone using F-127 as pore-forming agent, but the diameter of the microspheres was small, only used for sustained release of the drug, and F-127 was toxic, left much during mass production, toxic, poor in biocompatibility^[2]. Qutachi O, et al, used PBS as a pore-forming agent to prepare a porous PLGA scaffold and applied to the culture of 3T 3-mouse fibroblasts^[3]. However, PLGA is a hydrophobic material and can only be dissolved in organic solvents such as dichloromethane and acetone, and most of the pore-forming agents are water-soluble substances, and the oil phase and the water phase are mixed and then quickly delaminated, so that the obtained PLGA porous microspheres have non-uniform pores, and the large-scale industrial production is also difficult.

CN108348646A discloses a method for preparing porous polymer microspheres for preventing or treating soft tissue diseases, wherein gelatin is used as a pore-forming agent, and the obtained drug-loaded microspheres have non-uniform morphology. CN105982862A discloses a method for preparing porous microspheres, which uses the saturated vapor pressure difference of two solvents to perform pore-forming by electrostatic spinning, and finally obtains porous microspheres. Although the method uses a homogeneous system, the phenomenon of layering of a two-phase solvent is avoided, the requirement on production equipment is high, and the application range of the method is limited to a certain extent.

It has been reported that the size of microcarriers more suitable for cell culture is about 100-300 μm^[4,5]. In the currently reported manufacturing method for PLGA microspheres, PLGA is dissolved in a mixed solvent of dichloromethane and ethanol, then the drug is dissolved in a water phase with an emulsifier, then the drug is mixed with the PLGA solution for ultrasound, and finally the PVA solution is added for emulsification to obtain microspheres, even nanospheres, with a diameter of less than 10 μm. The mixed solvent can change the distribution form of the medicine and improve the medicine qualityThe entrapment efficiency is high, but the diameter of the sphere prepared by the method is small, most of the sphere is applied to drug slow release, and the sphere is not applied to cell culture and the like. PLGA microcapsules (microcapsules) prepared with ethanol as co-solvent in GravesR.A. 2006, with a diameter less than 100 μm, can increase enkephalin loading and slow release^[6](ii) a However, due to their small size, the PLGA microcapsules are still not suitable for cell proliferation.

Disclosure of Invention

Problems to be solved by the invention

In order to solve the problems that oil and water phases are easy to separate in the emulsification process of preparing microspheres, the internal structure of the microspheres is not uniform, and the microspheres are high in density, long in degradation time and the like, the PLGA is used as a raw material, the PLGA is dissolved in an organic solvent and then mixed with alcohol, and the PLGA microspheres which are hollow inside and have the diameter of 100-500 mu m are prepared by an emulsification method.

Means for solving the problems

In a first aspect, the present invention provides a method for preparing hollow polylactic acid-glycolic acid-gelatin composite microspheres, comprising the following steps:

1) dissolving polylactic acid-glycolic acid in an organic solvent to obtain a polylactic acid-glycolic acid solution;

2) adding a pore-foaming agent into the polylactic acid-glycolic acid solution obtained in the step 1), and homogenizing to form a mixed solution;

3) adding the mixed solution obtained in the step 2) into a polyvinyl alcohol aqueous solution below the liquid level, stirring to form an emulsion, and removing a pore-forming agent and an organic solvent to obtain a remainder;

4) collecting and washing the residues in the step 3) to obtain hollow polylactic acid-glycolic acid microspheres with the diameter of 100-500 mu m;

5) activating the surfaces of the hollow polylactic acid-glycolic acid microspheres obtained in the step 4) to obtain activated hollow polylactic acid-glycolic acid microspheres;

6) mixing and reacting the activated hollow polylactic acid-glycolic acid microspheres in the step 5) with a gelatin solution, and collecting, washing and drying to obtain the hollow polylactic acid-glycolic acid-gelatin composite microspheres.

Further, in the preparation method, the molar ratio of two monomer units in the polylactic acid-glycolic acid in the step 1), namely lactic acid and glycolic acid, is 85: 15-50: 50.

Further, in the preparation method, the weight average molecular weight of the polylactic acid-glycolic acid in the step 1) is 3000-200000 Da.

Further, in the above production method, the organic solvent in step 1) is at least one selected from the group consisting of dichloromethane, acetone, chloroform, ethyl acetate, hexane, petroleum ether, benzene, toluene, tetrahydrofuran, dimethyl sulfoxide and benzyl alcohol, preferably dichloromethane.

Further, in the preparation method, the mass concentration of the polylactic acid-glycolic acid solution in the step 1) is 1-35 g/100mL, preferably 2-10 g/100 mL.

Further, in the above preparation method, the porogen in step 2) is selected from at least one of methanol, ethanol, propanol, butanol and pentanol, preferably ethanol.

Further, in the preparation method, the volume ratio of the pore-forming agent to the organic solvent in the step 2) is 1: 1-1: 40, preferably 1: 2-1: 20.

Further, in the above production method, the homogenization in the step 2) is performed by using a homogenizer.

Further, in the above preparation method, the rotation speed of the homogenizer is 3000 to 30000rpm, preferably 10000 to 15000 rpm.

Further, in the above preparation method, the time for homogenization in step 2) is 5 to 600 seconds, preferably 30 to 100 seconds.

Further, in the above preparation method, the addition of the mixed solution in the step 3) is performed by injection.

Further, in the preparation method, the injection speed in the step 3) is 1-100 mL/min, preferably 10-20 mL/min.

Further, in the above production method, the mass concentration of the polyvinyl alcohol aqueous solution in the step 3) is 0.1 to 2g/100mL, preferably 0.5 to 1g/100 mL.

Further, in the preparation method, the volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution in the step 3) is 1:10 to 1:300, preferably 1:25 to 1: 100.

Further, in the above production method, the stirring in the step 3) is performed by a stirrer.

Further, in the above production method, the rotation speed of the stirrer is 30 to 600rpm, preferably 150 to 250 rpm.

Further, in the above preparation method, the molar ratio of NHS and EDC used for activation in step 5) (NHS: EDC) is 1:10 to 10:1, preferably 1:8 to 8:1, and more preferably 1:5 to 5: 1.

Further, in the preparation method, the activation time in the step 5) is 1-120 min, preferably 5-60 min, and more preferably 15-30 min.

Further, in the above preparation method, the gelatin solution in step 6) is a gelatin aqueous solution, wherein the mass concentration of gelatin (i.e., the mass of gelatin in 100mL of water) is 0.1 to 20%, preferably 0.5 to 10%, and more preferably 1 to 8%.

Further, in the above preparation method, the reaction between gelatin and the activated hollow PLGA microspheres in step 6) is performed under stirring; the reaction time is 1-24 h, preferably 4-18 h, and more preferably 6-12 h; the reaction temperature is 20-50 ℃, preferably 33-37 ℃.

In a second aspect, the present invention provides a hollow polylactic acid-glycolic acid-gelatin composite microsphere, which is prepared by the preparation method in the first aspect.

Further, the diameter of the polylactic acid-glycolic acid-gelatin composite microsphere is 100-500 μm, preferably 100-300 μm.

Further, the density of the polylactic acid-glycolic acid-gelatin composite microspheres is 0.9-1.4 g/cm³Preferably 1.0 to 1.1g/cm³。

In a third aspect, the invention provides a culture system and a culture method using the hollow polylactic acid-glycolic acid-gelatin composite microspheres as cell culture microcarriers.

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, the following technical effects can be obtained:

(1) by adding pore-foaming agents which are mutually soluble with the organic solvent and the water into an organic solvent-water system, the method disclosed by the invention overcomes the problem of oil-water layering which is very easy to occur in the emulsification process, and further avoids the problems of high density, slow degradation and the like of the prepared microspheres; the density is small, the diameter is 100-500 mu m, preferably 100-300 mu m, the micro-carrier meets the requirement of a three-dimensional cell culture micro-carrier, and the micro-carrier is suitable for cell culture;

(2) by regulating the dosage of the pore-forming agent, the method can also obtain microspheres with different wall thicknesses and diameters of 100-300 mu m, and the preparation process is simple and easy for industrial production;

(3) the carboxyl on the PLGA microspheres prepared by the method can be connected with biocompatible materials such as gelatin and the like, and the microspheres are hollow inside and have low density, so the PLGA microspheres are very suitable for being used as cell culture carriers and can be widely applied to the fields of tissue engineering and regenerative medicine.

Drawings

FIGS. 1A-1B show micrographs (100X) and SEM images of PLGA microspheres of example one.

Fig. 2 shows a micrograph (100 ×) of PLGA microspheres of example two.

FIGS. 3A-3B show SEM photographs of PLGA microspheres of example two.

FIGS. 4A-4B show micrographs (100X) and SEM images of PLGA microspheres of example three.

Fig. 5 shows a micrograph (100 x) of PLGA microspheres of example four.

FIGS. 6A-6B show micrographs (100X) and SEM images of PLGA microspheres of example five.

Fig. 7A-7B show micrographs (100 x) and SEM photographs of PLGA microspheres of example six.

FIGS. 8A-8B show micrographs (100X) and SEM images of PLGA microspheres of example seven.

FIGS. 9A-9B show fluorescence photographs of viable cell staining on PLGA-gelatin composite microspheres of example eleven.

Detailed Description

[ definition of terms ]

As used herein, unless otherwise indicated, "A to B" means a range of values inclusive of the A and B values as endpoints.

As used herein, unless otherwise specified, "may" or "may" mean to include performing a certain action, operation or process, or to exclude a corresponding action.

As used herein, unless otherwise specified, "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, mean that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

The term "microsphere" as used herein, unless otherwise indicated, refers to a polymeric microparticle that optionally adsorbs a drug. According to the difference of high molecular polymer matrix, it can be divided into natural high molecular microsphere (such as starch microsphere, gelatin microsphere, etc.) and synthetic high molecular microsphere (such as polylactic acid microsphere, polylactic acid-glycolic acid microsphere, etc.).

The term "polymerization" as used herein refers to a process of combining a small molecule compound into a high molecular compound by a polymerization reaction, and the "small molecule compound" as a reactant is generally referred to as a "monomer" and the "high molecular compound" as a product corresponding thereto is generally referred to as a "polymer", unless otherwise specified.

As used herein, unless otherwise indicated, the term "homogenization" (or "homogenizing") refers to the process of micronizing and homogenizing a dispersion in a suspension or emulsion system, which simultaneously reduces the size of the dispersion and improves the uniformity of distribution of the dispersion.

The term "injection" as used herein, unless otherwise indicated, refers to the process of quantitative transfer of a fluid by means of a syringe or like quantifiable fluid transfer device.

The term "emulsification" as used herein, unless otherwise specified, refers to the process of uniformly mixing two immiscible liquids. Essentially, emulsification is the uniform dispersion of one liquid in the form of tiny droplets (dispersed/internal) in another (continuous/external) by means of crushing and mixing. A common emulsification operation is mechanically forced emulsification (e.g. stirring).

As used herein, unless otherwise indicated, the term "agitation" refers to the process of uniformly dispersing a mixture of predominantly solid or liquid materials by rotating the mixture manually or by machine.

Hollow PLGA microspheres and preparation method thereof

The invention provides a preparation method of hollow PLGA microspheres. The method comprises the following steps:

1) dissolving PLGA in an organic solvent to obtain a PLGA solution;

2) adding a pore-foaming agent into the PLGA solution in the step 1), and homogenizing to form a mixed solution;

3) adding the mixed solution obtained in the step 2) into a PVA aqueous solution below the liquid level, stirring to form an emulsion, and removing a pore-forming agent and an organic solvent to obtain a remainder;

4) collecting and washing the residues in the step 3) to obtain hollow PLGA microspheres with the diameter of 100-500 mu m;

5) activating the surfaces of the hollow PLGA microspheres in the step 4) to obtain activated hollow PLGA microspheres;

6) mixing and reacting the activated hollow PLGA microspheres in the step 5) with a gelatin solution, and collecting, washing and drying to obtain the hollow PLGA-gelatin composite microspheres.

In some embodiments of the present invention, the PLGA in step 1) comprises two monomer units, Lactic Acid (LA) (or 2-hydroxypropionic acid) and Glycolic Acid (GA) (or glycolic acid, glycolic acid), which are polycondensed according to a certain molar ratio, the obtained PLGA has a certain molecular weight, and the end group can be carboxyl-terminated or ester-terminated.

In some preferred embodiments, the molar ratio of the two monomers-LA and GA in PLGA in step 1) is 85:15 to 50: 50; in other words, PLGA consists of 85% to 50% LA and 15% to 50% GA, the sum of which is 100%.

In some preferred embodiments, the weight average molecular weight of PLGA in step 1) is from 3000 to 200000 Da.

In some more preferred embodiments, the molar ratio of the two monomers-LA and GA in PLGA in step 1) is from 85:15 to 50:50, and the weight average molecular weight of PLGA is from 3000 to 200000 Da.

In the preparation method of PLGA microspheres by the emulsification method, it is necessary to use an organic solvent to dissolve PLGA as an oil phase in the emulsification method.

In some embodiments of the present invention, the organic solvent in step 1) is selected from at least one of Dichloromethane (DCM), Acetone (ACT), Chloroform (CLF), Ethyl Acetate (EA), Hexane (HEX), Petroleum Ether (PE), Benzene (BZN), Toluene (TOL), Tetrahydrofuran (THF), Dimethylsulfoxide (DMSO), and Benzyl Alcohol (BA).

In some preferred embodiments, the organic solvent in step 1) is DCM.

In some embodiments of the invention, the PLGA in step 1) needs to be maintained at a certain concentration in the organic solvent.

In some preferred embodiments, the mass concentration of the PLGA solution in the step 1) is 1-35 g/100 mL; in other words, the solution contains 1 to 35g of PLGA per 100mL of the solution.

In some more preferred embodiments, the mass concentration of the PLGA solution in step 1) is 2-10 g/100 mL.

In order to avoid oil-water two-phase separation in the emulsification process of preparing microspheres and further avoid the problems that the prepared microspheres are not uniform in structure and part of the microspheres are possibly overlarge in density and slow in degradation, the invention creatively adds alcohol serving as a pore-forming agent into an oil phase according to a certain proportion, forms a mixed solution after homogenization, then adds the mixed solution into a PVA aqueous solution, forms a multiple emulsion after stirring, and finally successfully prepares the PLGA microspheres with the diameters of 100-500 micrometers (preferably 100-300 micrometers) and hollow interiors through operations of solidification, washing, centrifugation and the like.

In some embodiments of the invention, the porogen in step 2) is selected from at least one of methanol, ethanol, propanol, butanol and pentanol.

In some preferred embodiments, the porogen in step 2) is ethanol.

In some more preferred embodiments, the porogen in step 2) is anhydrous ethanol.

In some embodiments of the present invention, the amount of porogen used in step 2) needs to match the amount of organic solvent used in step 1).

In some preferred embodiments, the volume ratio of the porogen in step 2) to the organic solvent in step 1) is 1:1 to 1: 40; in other words, 1 to 40mL of the organic solvent is used per 1mL of the porogen.

In some preferred embodiments, the volume ratio of the porogen in step 2) to the organic solvent in step 1) is 1:2 to 1: 20.

In some more preferred embodiments, the volume ratio of the porogen in step 2) to the organic solvent in step 1) is 1:2 to 1: 10.

Mixing between the porogen and the organic phase may be achieved by a homogenization operation. In some embodiments of the invention, the homogenization in step 2) is accomplished using a homogenizer. Controlling the rotation speed and working time of the homogenizer can accomplish a specific homogenization.

In some preferred embodiments, the rotation speed of the homogenizer in the step 2) is 3000 to 30000 rpm.

In some more preferred embodiments, the rotation speed when homogenizing in step 2) is carried out using a homogenizer is 10000 to 15000 rpm.

In some preferred embodiments, the time for homogenizing in step 2) is 5 to 600 seconds.

In some more preferred embodiments, the time for homogenizing in step 2) is 30 to 100 seconds using a homogenizer.

In some most preferred embodiments, the homogenizing in step 2) is performed using a homogenizer at 10000 to 15000rpm for 30 to 100 seconds.

At present, the PLA/PLGA type microspheres mostly adopt O/WThe type (or W/O/W type) preparation method^[7-10]The oil phase (or an oil phase comprising an internal aqueous phase) needs to be added to the aqueous phase (or an external aqueous phase) and is usually added (e.g. injected) in a quantitative or fixed rate. In some embodiments of the invention, the injection in step 3) is accomplished using a syringe pump.

In some preferred embodiments, the speed of the injection in step 3) is 1 to 100mL/min by using a syringe pump.

In some more preferred embodiments, the speed of the injection in step 3) is 10 to 20mL/min using a syringe pump.

In some preferred embodiments, the injection pump is used to perform step 3) and the injection outlet needle has an inner diameter of 0.10 to 0.50 mm.

In the preparation method of PLGA microspheres by the emulsification method, it is necessary to use water or a mixed solvent containing water to dissolve PVA as an aqueous phase in the emulsification method.

In some preferred embodiments, the mass concentration of the PVA aqueous solution in the step 3) is 0.1-2 g/100 mL; in other words, the PVA content is 0.1 to 2g per 100mL of the solution.

In some more preferred embodiments, the mass concentration of the PVA aqueous solution in the step 3) is 0.5-1 g/100 mL.

In the preparation method of PLGA microspheres by the emulsification method, the ratio of oil phase to water phase directly affects the final result.

In some preferred embodiments, the volume ratio of the organic solvent in the step 1) to the aqueous PVA solution in the step 3) is 1:10 to 1: 300; in other words, 10 to 300mL of the PVA aqueous solution is used per 1mL of the organic solvent.

In some preferred embodiments, the volume ratio of the organic solvent in step 1) to the aqueous PVA solution in step 3) is 1:25 to 1: 100.

Stirring is an effective means for realizing emulsification of oil and water phases. In some embodiments of the invention, the agitation in step 3) is accomplished using a blender. Agitators agitate the contents by rotation of an agitating member (e.g., paddle) in an agitator vessel are common methods for dispersing gas, liquid, or solid particles in a liquid.

The emulsification conditions have a great influence on the emulsification result, and one of the factors is the stirring speed. The proper stirring speed can fully mix the oil phase and the water phase, while the purpose of fully mixing cannot be achieved at the excessively low stirring speed, but bubbles are introduced into a stirring system at the excessively high stirring speed to form a three-phase system, so that the emulsion is unstable.

In some preferred embodiments, the rotation speed of the stirrer during stirring in the step 3) is 30-600 rpm.

In some more preferred embodiments, the rotation speed of the stirrer during stirring in step 3) is 150 to 250 rpm.

In some preferred embodiments, the removal of the organic solvent in step 3) is performed at normal temperature and pressure by volatilization.

In some preferred embodiments, the removal of the organic solvent in step 3) is performed by volatilization under vacuum at ambient temperature.

In some preferred embodiments, the water in step 4) is redistilled water, i.e. water that has undergone two distillations.

In some preferred embodiments, the number of washing in step 4) is 2 to 5.

In some preferred embodiments, the molar ratio of NHS and EDC used for activation in step 5) (NHS: EDC) is 1:8 to 8: 1.

In some more preferred embodiments, the molar ratio of NHS and EDC used for activation in step 5) (NHS: EDC) is 1:5 to 5: 1.

In some preferred embodiments, the activation time in step 5) is 1 to 120 min.

In some preferred embodiments, the time for activation in step 5) is 5 to 60 min.

In some more preferred embodiments, the time of activation in step 5) is 15 to 30 min.

In some preferred embodiments, the gelatin solution in the step 6) is a gelatin water solution, wherein the mass concentration of the gelatin is 0.1-20%; in other words, the gelatin is contained in an amount of 0.1 to 20g per 100mL of the solution.

In some more preferred embodiments, the mass concentration of gelatin in the gelatin solution in step 6) is 0.5-10%.

In some more preferred embodiments, the mass concentration of gelatin in the gelatin solution in step 6) is 1-8%.

In some preferred embodiments, the reaction between gelatin and activated hollow PLGA microspheres in step 6) is performed under stirring; the reaction time is 1-24 h, preferably 4-18 h, and more preferably 6-12 h; the reaction temperature is 20-50 ℃, preferably 33-37 ℃.

The invention also provides the hollow PLGA-gelatin composite microsphere. The hollow PLGA-gelatin composite microsphere is prepared by the preparation method.

In some embodiments of the present invention, the diameter of the hollow PLGA-gelatin composite microsphere is 100 to 500 μm.

In some preferred embodiments, the diameter of the hollow PLGA-gelatin composite microsphere is 100 to 300 μm.

In some embodiments of the present invention, the density of the hollow PLGA-gelatin composite microspheres is 0.9-1.4 g/cm³。

In some preferred embodiments, the density of the hollow PLGA-gelatin composite microspheres is 1.0 to 1.1g/cm³。

In some more preferred embodiments, the hollow PLGA-gelatin composite microspheres have a diameter of 100 to 300 μm and a density of 1.0 to 1.1g/cm³。

Application of hollow PLGA-gelatin composite microspheres

The hollow PLGA-gelatin composite microspheres prepared by the preparation method can be used for cell culture not only in a planar pore plate, but also in three-dimensional culture containers such as a shake flask, a bioreactor and the like. The hollow PLGA-gelatin composite microspheres have large surface area, hollow interior and small density, can improve the cell amplification efficiency, and can ensure the normal growth of stem cells particularly when the stem cells are cultured.

In the embodiment, the structure of the obtained microsphere can be adjusted by adjusting the using amount of the ethanol, and the pore diameter or the microsphere wall thickness can be adjusted. The thinner the microsphere wall, the lower the density of the microspheres and the faster the degradation rate. During three-dimensional culture, the microspheres can be stirred only at a low rotating speed, so that the shearing force on cells is reduced, and the damage and death of the cells are reduced. However, the wall of the microspheres cannot be too thin, and the excessively thin microspheres are not rigid enough and are easily broken during the cell culture stirring process, so that the surface area of the microspheres is reduced, and the cell culture efficiency is reduced. In one embodiment, the prepared hollow PLGA-gelatin composite microspheres can not be broken even if being subjected to a large shearing force during cell culture, and can be rapidly degraded after being injected into a body.

Therefore, the invention provides the application of the hollow PLGA-gelatin composite microsphere as a cell culture carrier.

The technical solution of the present invention will be further explained with reference to specific examples. It is to be understood that the following examples are only for illustrating and explaining the present invention and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, instruments, materials, reagents and the like used in the following examples are all available by conventional commercial means.

The first embodiment is as follows: and (4) preparing hollow PLGA microspheres.

PLGA (50:50, Mw 30000-50000 Da, carboxyl end-capped) (1g) was weighed and dissolved in dichloromethane (20 mL). Anhydrous ethanol (10mL) was added and homogenized by homogenizer for 60s at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And washing by redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figures 1A-1B.

The obtained hollow PLGA microspheres have a diameter of 100 μm and a density of 1.00-1.10g/cm³。

Example two: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (10mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And washing with redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figure 2.

As shown in FIGS. 3A-3B, the hollow PLGA microspheres prepared by the above method have a diameter of about 150 μm, uniform size distribution, smooth surface (1.50kX magnification), thin outer wall, hollow interior (400X magnification), and density of about 1.00-1.10g/cm³In the meantime.

Example three: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (10mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 120rpm for 12 hours to volatilize and remove methylene chloride. And washing by redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figures 4A-4B.

The PLGA microspheres prepared by the method have the diameter of about 200 μm, uniform size distribution, smooth surface, hollow interior and density of about 1.00-1.10g/cm³In the meantime.

Example four: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (10mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 5mL/min by means of a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And washing with redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figure 5.

The PLGA microspheres prepared by the above method have a diameter of about 250 μm and a size of about 250 μmThe cloth is uniform, the surface is smooth, the interior is hollow, and the density is about 1.00-1.10g/cm³In the meantime.

Example five: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end capped) (1g) was weighed, dissolved in dichloromethane (20mL), added with absolute ethanol (6.66mL), and homogenized in a homogenizer for 60s at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And washing by redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figures 6A-6B.

The PLGA microspheres prepared by the method have the diameter of about 200 μm, smooth surface, hollow interior and density of about 1.00-1.10g/cm³But the outer wall of the microsphere is thickened to a greater extent than the wall thickness of the microsphere of example two.

Example six: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (5mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. Washing with redistilled water for 3 times, centrifuging and collecting to obtain hollow PLGA microspheres shown in figures 7A-7B.

The PLGA microspheres prepared by the method have the diameter of about 250 μm, smooth surface, hollow interior and density of about 1.00-1.10g/cm³And the outer wall of the microsphere is further thickened, and the wall thickness is larger than that of the microsphere in the fifth embodiment.

Example seven: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (2mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And washing by redistilled water for 3 times, centrifuging and collecting to obtain the hollow PLGA microspheres shown in figures 8A-8B.

The PLGA microspheres prepared by the method have the diameter of about 300 mu m, smooth surface and density of about 1.00-1.10g/cm³The inner hollow structure is further reduced, and the wall thickness is larger than that of the microsphere in the sixth embodiment.

As can be seen from comparison between the second and fifth to seventh examples, the hollow structure of the PLGA microspheres obtained was substantially reduced with the decrease in the amount of ethanol (the volume ratio of the absolute ethanol to the dichloromethane was 1:2, 1:3, 1:4 and 1:10), indicating that ethanol indeed functions as a porogen. And the internal structure of the microsphere can be adjusted by adjusting the using amount of ethanol, so that the required hollow PLGA microsphere is obtained.

Example eight: and (4) preparing hollow PLGA microspheres.

PLGA (75:25, Mw 66000-105000 Da, ester group end-capped) (1g) was weighed, dissolved in dichloromethane (20mL), and added with absolute ethanol (1mL), homogenized for 60s with a homogenizer at 13000 rpm. The mixed solution was injected into a 0.5% PVA aqueous solution (2L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, and stirred at a mechanical stirring rate of 200rpm for 12 hours to volatilize and remove methylene chloride. And (4) washing by redistilled water for 3 times, and centrifugally collecting to obtain the hollow PLGA microspheres.

Example nine: and (4) preparing hollow PLGA microspheres.

PLGA (85:15, Mw 50000-120000 Da, ester group end capping) (1g) was weighed, dissolved in dichloromethane (10mL), and added with absolute ethanol (5mL), homogenized for 100s with a homogenizer at 13000 rpm. The mixed solution was injected into a 1% PVA aqueous solution (1L) at a rate of 10mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, mechanically stirred at 250rpm for 12 hours, and methylene chloride was evaporated. And (4) washing by redistilled water for 3 times, and centrifugally collecting to obtain the hollow PLGA microspheres.

Example ten: and (4) preparing hollow PLGA microspheres.

PLGA (85:15, Mw 50000-120000 Da, ester group end capping) (1g) was weighed, dissolved in dichloromethane (10mL), and added with absolute ethanol (0.5mL), homogenized for 100s with a homogenizer at 13000 rpm. The mixed solution was injected into a 1% PVA aqueous solution (1L) at a rate of 20mL/min by a syringe pump equipped with a 25G syringe needle having an inner diameter of 0.25mm, mechanically stirred at 200rpm for 12 hours, and methylene chloride was evaporated. And (4) washing by redistilled water for 3 times, and centrifugally collecting to obtain the hollow PLGA microspheres.

Example eleven: and (3) preparing the hollow PLGA-gelatin composite microspheres.

The hollow PLGA microspheres (1g) prepared in example one were soaked in an aqueous solution (50mL) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (8mM) and N-hydroxysuccinimide (NHS) (2.66mM) and the carboxyl groups were activated for 15min using a decolorizing shaker (200 rpm). And (4) washing by redistilled water for 3 times, centrifuging and collecting to obtain the carboxyl activated hollow PLGA microspheres.

Adjusting the pH value of a gelatin aqueous solution (40mL) with the mass concentration of 4% to 8.0-9.0 by using a 1M sodium hydroxide aqueous solution, then adding the gelatin aqueous solution into the hollow PLGA microspheres activated by carboxyl, oscillating the mixture overnight in a shaking table under the conditions of 200rpm and 35 ℃, repeatedly washing the mixture by redistilled water the next day, and centrifugally collecting the mixture to obtain the hollow PLGA-gelatin composite microspheres.

And measuring the content of the gelatin in the hollow PLGA-gelatin composite microspheres by using a BCA method. The method comprises the following operation steps: weighing 50mg of hollow PLGA-gelatin composite microspheres, dissolving the hollow PLGA-gelatin composite microspheres in 0.5mL of dimethyl sulfoxide solution completely, and diluting the hollow PLGA-gelatin composite microspheres by 10 times with redistilled water to obtain a sample solution 1; the same treatment method was used for the hollow PLGA base microspheres as solution 2, which was used as the background during detection. A range of concentrations of gelatin were dissolved in dimethylsulfoxide: water-1: 9(v/v) mixed solution, used to establish a standard curve. The above solutions were mixed with BCA working solution at 25 μ L: mixing at the ratio of 200 μ L, incubating in an incubator at 37 deg.C for 30min, detecting the absorbance, subtracting the absorbance of the sample solution 2 from the absorbance of the sample solution 1, and obtaining the gelatin concentration according to the standard curve of the gelatin solution. According to the method, the mass content of the gelatin in the hollow PLGA-gelatin composite microspheres is 4%.

Example twelve: and (3) culturing cells by using the hollow PLGA-gelatin composite microspheres.

Weighing the hollow PLGA-gelatin composite microspheres (0.1g) prepared in EXAMPLE eleventh, sterilizing, rinsing with 50mL Phosphate Buffered Saline (PBS) thoroughly, placing into a cell culture roller bottle, adding a stem cell culture medium (Tianjin tertiary thing) (50mL), placing into a cell culture box, and culturing in 5% CO₂And pre-culturing at 37 ℃ at a rotation speed of 35rpm so that the microspheres can be uniformly suspended in the spinner flask. Inoculating stem cells after preculture for 24h, wherein the inoculation amount of the cells is 1 multiplied by 10⁵Each cell was left to stand for 30min every 5min with stirring, and 6 cycles were repeated in total, and the cells were cultured in a suspended state, and were stained with FDA on the next day, and the adhesion and proliferation of the cells were observed, and the results are shown in FIGS. 9A to 9B.

As shown in FIGS. 9A-9B, after 24h of spinner culture, the fluorescence emitted after stem cell staining can be observed, which indicates that the hollow PLGA-gelatin microspheres have good adhesion to stem cells and can be used as cell culture carriers to increase the cell proliferation multiple.

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Claims

1. a preparation method of hollow polylactic acid-glycolic acid microspheres comprises the following steps:

2) adding a pore-foaming agent into the polylactic acid-glycolic acid solution, and homogenizing to form a mixed solution;

3) adding the mixed solution below the liquid level into a polyvinyl alcohol aqueous solution, stirring to form an emulsion, and removing the pore-forming agent and the organic solvent to obtain a remainder;

4) and collecting and washing the residues to obtain the hollow polylactic acid-glycolic acid microspheres with the diameter of 100-500 mu m.

2. The method according to claim 1, wherein the molar ratio of the lactic acid monomer unit to the glycolic acid monomer unit in the polylactic acid-glycolic acid is 85:15 to 50:50, and the weight average molecular weight of the polylactic acid-glycolic acid is 3000 to 200000 Da.

3. The production method according to claim 1, wherein the organic solvent is at least one selected from the group consisting of dichloromethane, acetone, chloroform, ethyl acetate, hexane, petroleum ether, benzene, toluene, tetrahydrofuran, dimethyl sulfoxide, and benzyl alcohol; the pore-foaming agent is selected from at least one of methanol, ethanol, propanol, butanol and pentanol.

4. The preparation method according to claim 1, wherein the organic solvent is dichloromethane and the porogen is ethanol.

5. The method according to claim 1, wherein the mass concentration of the polylactic acid-glycolic acid solution is 1 to 35g/100 mL.

6. The preparation method according to claim 1, wherein the volume ratio of the porogen to the organic solvent is 1: 1-1: 40.

7. The preparation method according to claim 1, wherein the volume ratio of the porogen to the organic solvent is 1: 2-1: 20.

8. The preparation method according to claim 1, wherein the homogenizing is performed by a homogenizer, and the rotation speed of the homogenizer is 3000-30000 rpm; the homogenizing time is 5-600 s.

9. The preparation method according to claim 1, wherein the mixed solution is added to the aqueous polyvinyl alcohol solution by injection, wherein the injection speed is 1-100 mL/min; the rotating speed of the stirring is 30-600 rpm.

10. The preparation method according to claim 1, wherein the mass concentration of the polyvinyl alcohol aqueous solution is 0.1-2 g/100 mL; the volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution is 1: 10-1: 300.

11. A preparation method of hollow polylactic acid-glycolic acid-gelatin composite microspheres comprises the following steps:

1) activating the surface of the hollow polylactic acid-glycolic acid microsphere of claim 1 to obtain an activated hollow polylactic acid-glycolic acid microsphere;

2) mixing and reacting the activated hollow polylactic acid-glycolic acid microspheres with a gelatin solution, collecting, washing and drying to obtain hollow polylactic acid-glycolic acid-gelatin composite microspheres, wherein the surfaces of the composite microspheres are combined with gelatin through covalent bonds, the mass of the gelatin is less than 10% of the total mass of the composite microspheres, and the diameter of the composite microspheres is 100-500 microns.

12. The method according to claim 11, wherein the molar ratio of NHS to EDC used for the activation is 1:10 to 10: 1.

13. The method according to claim 11, wherein the activation time is 1 to 120 min.

14. The preparation method according to claim 11, wherein the gelatin solution is a gelatin aqueous solution, and the mass concentration of gelatin is 0.1-20%.

15. The preparation method according to claim 11, wherein the reaction is carried out under stirring for 1 to 24 hours at a temperature of 20 to 50 ℃.

16. Hollow polylactic acid-glycolic acid microspheres obtained by the method of preparation according to any one of claims 1 to 10.

17. A hollow polylactic acid-glycolic acid-gelatin composite microsphere prepared by the preparation method according to any one of claims 11 to 15.

18. The hollow polylactic acid-glycolic acid-gelatin composite microsphere according to claim 17, wherein the density of the hollow polylactic acid-glycolic acid-gelatin composite microsphere is 1.0-1.1 g/cm³。

19. A method for culturing cells, which comprises culturing cells using the hollow polylactic acid-glycolic acid microspheres according to claim 16 or the hollow polylactic acid-glycolic acid-gelatin composite microspheres according to claim 17 as a microcarrier.

20. The method of claim 19, wherein the cells are adherent cells.

21. The method of claim 20, wherein the adherent cells are 293, HEK-293T, 293TN, 293FT, AAV-293, HUVEC, ECV-304, L929, WB-F344, L-02, THP-1, D407, CHO, mesenchymal stem cells, embryonic stem cells, or IPS.