preparation of porous polylactide microspheres by emulsion-solvent evaporation based on solution...

POLYMERS FOR ADVANCED TECHNOLOGIES

Polym. Adv. Technol. 2005; 16: 622–627

Published online 24 June 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pat.629

Preparation of porous polylactide microspheres by

emulsion-solvent evaporation based on solution

induced phase separation

Yi Hong, Changyou Gao*, Yanchao Shi and Jiacong ShenDepartment of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

Received 9 December 2004; Revised 11 April 2005; Accepted 20 April 2005

Porous polylactide (PLA) microspheres were fabricated by an emulsion-solvent evaporation meth-

od based on solution induced phase separation. Scanning electron microscopy (SEM) observations

confirmed the porous structure of the microspheres with good connectivity. The pore size was in

the range of decade micrometers. Besides large cavities as similarly existed on non-porous micro-

spheres, small pores were found on surfaces of the porous microspheres. The apparent density of

the porous microspheres was much smaller than that of non-porous microspheres. Fabrication

conditions such as stirring rate, good solvent/non-solvent ratio, PLA concentration and dispersant

(polyvinyl alcohol, PVA) concentration had an important influence on both the particle size and

size distribution and the pore size within the microspheres. A larger pore size was achieved at a

slower stirring rate, lower good solvent/non-solvent ratio or lower PLA concentration due to longer

coalescence time. Copyright # 2005 John Wiley & Sons, Ltd.

KEYWORDS: polylactide; porous microspheres; phase separation; structure; density

INTRODUCTION

Polylactide (PLA) has been shown to be important serving as

tissue engineering scaffolds and drug delivery carriers

because of its good mechanical properties, non-toxicity, pro-

cessibility, and biodegradability. As a biomedical material,

PLA can be processed into solid products such as rods,1

bone screws,2 suture lines, drug carriers3 and microspheres,4

as well as porous structure such as scaffolds,5 non-woven fab-

rics6 and hollow microspheres.7 Among which the micro-

spheres are of particular interest because of their special

structure and tailorable properties. They have been, for

example, used as delivery vehicles for drugs, polypeptides,

proteins and genes.8–11 In the tissue engineering field, the

microspheres can also serve as microcarriers for cell

culture, having the unique feature that they can be directly

injected into the defect.12–14 Moreover, they can be further

assembled into a three-dimensional (3D) porous scaffold

either in vitro or in vivo to induce cell infiltration and tissue

regeneration.15

Some existing methods, such as phase separation,16

emulsion-solvent evaporation,9,10 spray drying17,18 have

been employed to prepare non-porous microspheres. Among

which the emulsion-solvent evaporation is frequently

adopted. In a typical procedure, a volatile organic phase

containing dissolved polymers is emulsified in an aqueous

phase at a continuous constant stirring rate. Then the volatile

solvent is evaporated gradually to form the spherical

polymer particles, which are generally non-porous inside.

However, microspheres with a porous structure in their

interior are much more appreciated than the non-porous ones

in many cases. For example, because of their large size and

low mass density, porous microspheres can be aerosolized

more easily than smaller non-porous microspheres, resulting

in higher respirable fractions of inhaled therapeutics. With

larger size, they can avoid phagocytic clearance from the

lungs before the therapeutic dose is delivered. These features

are particularly useful for controlled-release inhalation

therapy.19 However, dramatic reduction of acidic degrada-

tion products, mainly the lactic acid, can be achieved while

keeping the overall volume constant. This would be

extremely important for the spheres used as scaffolds in

tissue engineering, and injectable carriers in drug deliv-

ery.8,13,15 Concentrated acids surrounding the implant sites

will easily cause non-infected inflammation, and even tissue

necrosis. But few works have been reported on fabrication of

porous microspheres, which contain both tiny pores and

polymer matrix (the pore walls) throughout the sphere

bodies. Rapid solvent removal,20 porogen leaching12 and

double-emulsification19,21 have been used to fabricate porous

microspheres based on emulsion-solvent evaporation

method. Yet the procedures of these methods are rather

complicated for repeatable production and subtly operation.

Solution induced phase separation (SIPS) is a simple way to

obtain materials with porous structure. A typical system

contains polymers, good solvent and non-solvent. These two

kinds of solvents are miscible with each other. Along with an

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: C. Gao, Department of Polymer Science andEngineering, Zhejiang University, Hangzhou 310027, China.E-mail: [email protected]

increase of the non-solvent and the polymer concentration,

the polymer-rich phase will separate from the polymer-lean

phase. The porous structure is thus formed after removal of

the solvents. In principle, it is very similar to the traditional

emulsion-solvent evaporation process, except for the exis-

tence of the non-solvent. This feature makes the process

contain the simplicity, convenience and effectiveness of the

traditional emulsion-solvent evaporation, while porous

microsphere can be produced.

In this paper the fabrication of porous PLA microspheres

combining the methods of emulsion-solvent evaporation and

SIPS are reported. Scanning electron microscopy (SEM) is

employed to visualize the porous nature of the resultant

microspheres. The method introduced here could also be

extended to other soluble polymers. The fabricated porous

PLA microspheres may find diverse applications as drug

delivery carriers, tissue engineering scaffolds, cell carriers

and adsorption matrices, etc.

EXPERIMENTAL

MaterialsPLA (average Mw¼ 200 000) was synthesized according to

the method described previously.22 Methylene chloride

(CH2Cl2), n-hexane and poly(vinyl alcohol) 124 (PVA 124,

average Mw 85 000–124 000, 98–99% hydrolyzed) are all of

analytical grade and used as received.

Preparation of porous PLA microspheresIn a typical fabrication process, 0.5 g PLA was dissolved in

9 ml methylene chloride. Then 1 ml n-hexane was added

into the solution. The transparent 5% PLA/CH2Cl2-hexane

solution (oil phase) was poured into 100 ml water (water

phase) containing suitable amount of PVA dispersant. The

oil to water ratio was kept at 1:10. The system was stirred at

a constant rate for 24 hr at 258C to evaporate the organic sol-

vent. After collected by filtration and washed with deionized

water for five times at reduced pressure, the microspheres

were dried at 358C for 3 days. Detailed formulations are

described in Table 1. All the yields were higher than 85%.

Size distribution and number average diameterThe microspheres were sieved by a series of stainless steel

meshes and weighted. Number average diameter of the

microspheres was calculated according to eqn (1).

Dn ¼X DiWi=½4�ðDi=2Þ3�=3�

PWi=½4�ðDi=2Þ3�=3�

ð1Þ

where Dn represents number average diameter, Wi repre-

sents weight of the microspheres at i size, and Di represents

average diameter at i size. r represents the density of the

microspheres, which is regarded as a constant for the same

batch of products.

Apparent density of the porous microspheresA known volume (V) of the vessel was filled with the dried

porous microspheres and weighed (W, three parallel mea-

surements were taken). The apparent density (ra) was calcu-

lated according to eqn (2) under the hypothesis that the size of

the porous spheres was the same.

�a ¼W

V � Cð2Þ

where C, 0.601, is the theoretical value of a 3D loose random

packing density of spheres.23 Introduction of this constant

can make the apparent density closer to the real density

of the porous microsphere because the packing holes can

be ruled out.

Scanning electron microscopy (SEM)Scanning electron micrographs were taken on a JSM 5000

after being treated under reduced pressure for 3 days and

coated with gold. A cross-section of the microspheres was

fabricated by imbedding the microspheres in aqueous paraf-

fin. After complete solidification, the hardened paraffin was

then cut into pieces with a blade. Hemispheres imbedded in

the paraffin were extensively washed with n-hexane. The

average diameter of the inner pores was measured by SMILE

VIEW software supplied by JEOL Company.

RESULTS AND DISCUSSION

Scheme 1 presents the microstructure alteration in the fabri-

cation process of the porous PLA microsphere. In this system,

methylene chloride and n-hexane serve as a good solvent and

a non-solvent for PLA, respectively. They are also miscible

with each other. The PLA solution dissolved in the mixed

solvents is poured into a continuously stirred PVA aqueous

solution. The oil phase is subsequently dispersed into a

large number of small droplets under agitation. Though

both the organic solvents are volatile, the evaporation speed

of methylene chloride, however, is faster than that of n-hex-

ane because of its lower boiling point (40.48C). Therefore, the

ratio between methylene chloride and n-hexane will decrease

gradually along with the evaporation process, accompanying

Table 1. Properties of the porous microspheres as a function of fabrication conditionsa

SampleCPLA

(w/v)CPVA

(w/v)Stirring rate

(rpm)Solvent/

non-solventDn

(mm)Yield(%)

ra

(g/cm3)Inner pore

(mm)

A 5% 0.8% 300 9:1 471 98.5 0.263 24� 4B 5% 0.8% 500 9:1 290 98.7 0.356 16� 3C 5% 0.8% 800 9:1 176 97.3 0.386 12� 2D 5% 0.8% 500 9.5:0.5 251 86 0.475 5� 1E 5% 0.8% 500 8:2 413 87 0.213 30� 5F 5% 0.5% 500 9:1 390 99.7 0.306 10� 2G 3% 0.8% 500 9:1 241 99.2 0.246 20� 4H 8% 0.8% 500 9:1 429 87.9 0.395 7� 2

aOil/water ratio was 1:10 and evaporation temperature was 258C.

Copyright # 2005 John Wiley & Sons, Ltd. Polym. Adv. Technol. 2005; 16: 622–627

Preparation of porous polylactide microspheres 623

with increase of the PLA concentration. When the PLA con-

centration and the good solvent/non-solvent ratio reach a cri-

tical point, phase separation will occur. Along with the

further solvent evaporation, the polymer-lean phase will

spread and coalesce, resulting in size increase of a single

phase which corresponds to the tiny pores after complete sol-

vent removal. Finally, the polymer-lean phase will form the

pores surrounded by the polymer matrix formed from the

polymer-rich phase. Apparently, the phase separation occurs

mainly in the droplets, hence has no or at least minimal influ-

ence on the fabrication process, and on other properties of the

resultant microspheres such as size and size distribution.

All the microspheres are spherical as shown in Fig. 1. The

porous PLA microspheres possessed a rough surface with

many cavities and small pores. By contrast, only large cavities

were observed on the microspheres without addition of

Solvent evaporation

Phase spreadin droplet

Complete solventevaporation

Polymer-lean phase

porous PLAmicrosphere

Pore

PVA aqueoussolution

PLA+CH2Cl2+Hexane

PLA solutiondroplet

Solvent evaporation

Phase separationin droplet

Polymerrich phase

Hexane/CH2Cl2 ratio

PLA concentration

Coalescence

Scheme 1. Illustration to show the preparation and microstructure alteration processes of

porous PLA microspheres.

(a) (b)

500µm 100µm

100µm

(c)

100µm

Figure 1. SEM images to show the surface morphologies of microspheres. (a)

Sample A, (b) sample A with higher magnification (see Table 1 for sample

designation), (c) non-porous microspheres.

624 Y. Hong et al.


the non-solvent, n-hexane (Fig. 1c). No small pores could be

detected in this case. The other distinct difference is that the

porous microspheres float on water while the non-porous

microspheres sedimentate under water. The reason for the

formation of the big cavities is not quite clear at present.

Solvent evaporation could be basically ruled out since

evaporation at higher temperature (for example 608C) has

yielded porous microspheres with same surface morphol-

ogy. A possible reason could be that the shear stress

may create defects during the solvent evaporation process.

The small pores on the porous microspheres, however, are

attributed to the removal of the non-solvent, i.e. the polymer-

lean phase.

SEM observations reveal the porous structure in the

interiors of the PLA microspheres, as exemplified here with

three typical samples (Fig. 2). The irregular pores connect

with each other and homogenously distribute throughout the

whole spheres. This nice connecting other than close pore

structure would be favorable for loading of substances in the

case required. The inner pore size also was variable by

fabrication conditions as presented later. It is worth noting

that the paraffin imbedding is a very useful technique to

maintain the initial microstructure of the sliced micro-

spheres.

To explore the controlling factors for the microstructure of

the porous PLA microspheres, the polymer concentration,

the dispersant concentration, the stirring rate and the good

solvent/non-solvent ratio were varied as listed in Table 1.

Stirring rateKeeping other conditions constant, the influence of stirring

rate was firstly investigated with respect to particle size, par-

ticle size distribution, apparent density and inner pore size

(Table 1 sample A, B and C, Figs. 2 and 3). Along with the

increase in the stirring rate from 300 to 800 rpm, the number

average diameter of the particles was decreased from 470 to

180 mm, respectively (Table 1). All the particle size distribu-

tions were quite narrow and accordingly shifted to the smal-

ler size region (Fig. 3a–3c). By contrast, the apparent density

of the microspheres was increased accompanying with the

reduction of the inner pore size of the microspheres (Table 1

and Fig. 2).

At a higher stirring rate, a larger shear energy is supplied.

Hence larger surface area can be equilibrated, which

corresponds to smaller droplets and particle size.24 Theore-

tically, the apparent density can characterize the change of

porosity of the porous PLA microspheres, i.e. a lower density

should correspond to a higher porosity. From this point of

view, the porosity of the microspheres should decrease as a

function of the stirring rate, or the particle size. However this

is not definite since the porosity is theoretically decided by

the polymer concentration assuming that the extent of the

volume shrinkage is constant for all the systems during

solvent removal. The particle size and size distribution may

also influence the detection since the 3D loose random

packing density can be altered in practice, e.g. smaller than

the proposed value, 0.601. To testify this influence and to

compare the apparent density of porous particles with non-

porous ones, non-porous microspheres with diameters

ranging 100–154, 154–180, 280–450 and 450–710 mm were

similarly characterized, resulting in apparent densities of

1.124, 1.113, 1.083 and 1.049 g/cm3, respectively. These data

indeed demonstrated that the apparent density of the non-

porous spheres was much bigger than that of the porous ones,

(c)

500µm 50µm

50µm 50µm

(a) (b)

(d)

Figure 2. SEM images to show the inner microstructure of the porous

microspheres. (a) Sample A, (b) sample A with higher magnification, (c) sample

B, and (d) sample E.



and decreased as a function of particle size as well. These

results reveal also that the packing density of the measured

spheres is smaller than 0.601. Therefore, it is not clear at

present the exact correlation between the stirring rate and the

porosity. From those results, however, one can still conclude

that the influence of the stirring rate on the porosity should be

minimal.

As to the microstructure inside the porous spheres, a

higher stirring rate resulted in smaller pore size as shown in

Fig. 2(b) and 2(c) and Table 1. This is understood as a result of

faster solvent evaporating speed since the size of the droplets

is smaller. In the phase separation process, a faster solvent

removal would mean that the phase has a shorter coalescence

time, thus limiting the formation of larger phase domains.

Good solvent/non-solvent ratioGood solvent/non-solvent ratio is another key factor control-

ling properties of the porous microspheres. As the ratio

decreases, the solubility of polymers shall decrease, leading

to an accelerated phase separation process. As a result, the

separated phase domains would have longer time to grow

and coalesce with each other, thus larger pore size can be pro-

duced. As expected, when the ratio of the good solvent/poor

solvent decreased from 9.5:0.5 to 8:2, the pore size increased

from 5 to 30 mm accordingly as shown in Fig. 2 and Table 1

(sample B, D and E). The microspheres are solidified at a

much earlier stage during the solvent evaporation process

at a higher poor solvent ratio, leading to a smaller shrinkage

extent of the oil droplets (the shrinkage of the oil droplets in

solvent evaporation should always occur in the emulsion-

solvent evaporation method). Therefore, a larger particle

size was yielded as shown in Fig. 3(d) and Table 1. This

will, of course generate a lower density or higher porosity

of the microspheres, as illustrated in Table 1.

PLA concentrationThe concentration of polymers in the mixed solvents has

shown a proportional relationship with the particle size

and the apparent density (Table 1, sample G, B and H). The

viscosity of the system shall be increased at a higher polymer

concentration, leading to the dispersion difficulty. Hence

larger oil droplets will be produced. As such one can predict

that a higher polymer concentration would generate a

similar effect with slower stirring rate with respect to

particle size. Considering the relationship between the

particle size and the apparent density, one can find that the

0

10

20

30

40

50

60

>710

µm

450-

710µ

m

280-

450µ

m

180-

280µ

m

154-

180µ

m0

10

20

30

40

50

60

70

450-

710µ

m

>710

µm

280-

450µ

m

180-

280µ

m

154-

180µ

m

0

10

20

30

40

50

>710µm

450-

710µm

280-

450µm

180-

280µm

154-

180µm

100-

154µm

Wei

ghtr

atio

(%)

0

10

20

30

40

50

60

70

>710

µm

450-

710µm

280-

450µm

180-

280µm

Wei

ghtr

atio

(%)

(a)

(c) (d)

(b)

Wei

ghtr

atio

(%)

Wei

ghtr

atio

(%)

Figure 3. Particle size distribution histograms of the porous microspheres. (a) Sample A, (b) sample B, (c) sample C, and (d)

sample E.

626 Y. Hong et al.


polymer concentration has exactly the reverse effect with the

stirring rate. Therefore, the higher apparent density at a

higher polymer concentration is understood as a result of a

larger mass existing in the microspheres, which also means

a smaller porosity. Regarding the pore size, the PLA concen-

tration has shown a reverse effect with the good solvent/non-

solvent ratio. As shown in Table 1, the pore size decreased

from 20� 4 to 7� 2 mm when the PLA concentration was

improved from 3 to 8%. This should be mainly caused by

the viscosity increase, which slows down the coalescence

after phase separation.

PVA concentrationThe oil droplets formed under agitation are stabilized by the

dispersant added in the system, being the PVA in the present

case. Table 1 (samples B and F) shows that a higher PVA

concentration yielded a smaller particle size but larger appar-

ent density and pore size. It is well understood that a higher

concentration of dispersant is beneficial to decrease the inter-

facial energy of oil droplets, thus smaller droplets that cor-

respond to larger surface area can be formed. As discussed

earlier, the apparent density increase would not mean a por-

osity decrease in the present case. With higher PVA concen-

tration, diffusion of the organic solvents might become

harder. This will likely prolong the coarsening time, hence

a larger pore size is achieved.

CONCLUSIONS

In this study porous PLA microspheres have been success-

fully prepared by the emulsion-solvent evaporation method

based on solution induced phase separation. Their porous

nature inside the spheres is verified by SEM observations.

These irregular pores are in the range of decade micrometers.

They are well interconnected and homogeneously distributed

throughout the entire microspheres. The apparent density of

the porous microspheres, from 0.20 to 0.45 g/cm3, was much

smaller than that of the non-porous microspheres (>1 g/cm3).

A slower stirring rate, lower good solvent/non-solvent ratio

or lower PLA concentration is helpful to achieve larger pore

size. Together with dispersant concentration, the particle

size is modulated from 180 to 470mm. Moreover, these fabri-

cation factors can also influence the apparent density more or

less either by size variance or porosity alteration or both.

AcknowledgementsThis study is financially supported by the Natural Science

Foundation of China (Nos 20434030 and 90206006) and the

National Science Fund for Distinguished Young Scholars of

China (No. 50425311).

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