Preparation of porous polylactide microspheres by emulsion-solvent evaporation based on solution induced phase separation

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1 POLYMERS FOR ADVANCED TECHNOLOGIES Polym. Adv. Technol. 5; 16: Published online 24 June 5 in Wiley InterScience ( DOI: 1.12/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 Shen Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 3127, China Received 9 December 4; Revised 11 April 5; Accepted April 5 Porous polylactide (PLA) microspheres were fabricated by an emulsion-solvent evaporation method 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 microspheres, 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 # 5 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, processibility, and biodegradability. As a biomedical material, PLA can be processed into solid products such as rods, 1 bone screws, 2 suture lines, drug carriers 3 and microspheres, 4 as well as porous structure such as scaffolds, 5 non-woven fabrics 6 and hollow microspheres. 7 Among which the microspheres 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 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 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, 9,1 spray drying 17,18 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 *Correspondence to: C. Gao, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 3127, China. cygao@mail.hz.zj.cn 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 degradation 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 delivery. 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, porogen leaching 12 and double-emulsification 19,21 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 # 5 John Wiley & Sons, Ltd.

2 Preparation of porous polylactide microspheres 623 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 existence 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 Materials PLA (average M w ¼ ) was synthesized according to the method described previously. 22 Methylene chloride (CH 2 Cl 2 ), n-hexane and poly(vinyl alcohol) 124 (PVA 124, average M w , 98 99% hydrolyzed) are all of analytical grade and used as received. Preparation of porous PLA microspheres In a typical fabrication process,.5 g PLA was dissolved in 9 ml methylene chloride. Then 1 ml n-hexane was added into the solution. The transparent 5% PLA/CH 2 Cl 2 -hexane solution (oil phase) was poured into 1 ml water (water phase) containing suitable amount of PVA dispersant. The oil to water ratio was kept at 1:1. The system was stirred at a constant rate for 24 hr at 258C to evaporate the organic solvent. 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 diameter The 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 ¼ X DiWi=½4ðDi=2Þ 3 =3Š P Wi=½4ðDi=2Þ 3 =3Š ð1þ where Dn represents number average diameter, Wi represents 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 microspheres A known volume (V) of the vessel was filled with the dried porous microspheres and weighed (W, three parallel measurements were taken). The apparent density (r a ) was calculated according to eqn (2) under the hypothesis that the size of the porous spheres was the same. a ¼ W ð2þ V C where C,.61, 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 5 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 paraffin. 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 fabrication 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-hexane because of its lower boiling point (4.48C). 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 conditions a Sample C PLA (w/v) C PVA (w/v) Stirring rate (rpm) Solvent/ non-solvent Dn (mm) Yield (%) r a (g/cm 3 ) Inner pore (mm) A 5%.8% 9: B 5%.8% 5 9: C 5%.8% 8 9: D 5%.8% 5 9.5: E 5%.8% 5 8: F 5%.5% 5 9: G 3%.8% 5 9: H 8%.8% 5 9: a Oil/water ratio was 1:1 and evaporation temperature was 258C. Copyright # 5 John Wiley & Sons, Ltd. Polym. Adv. Technol. 5; 16:

3 624 Y. Hong et al. PLA+CH2Cl2 +Hexane Polymer rich phase Solvent evaporation PVA aqueous solution Hexane/CH2Cl2 ratio PLA concentration Phase separation in droplet PLA solution droplet Polymerlean phase Pore Complete solvent evaporation Solvent evaporation Coalescence porous PLA microsphere Phase spread in droplet Scheme 1. Illustration to show the preparation and microstructure alteration processes of porous PLA microspheres. with increase of the PLA concentration. When the PLA concentration and the good solvent/non-solvent ratio reach a critical 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 solvent removal. Finally, the polymer-lean phase will form the pores surrounded by the polymer matrix formed from the (a) polymer-rich phase. Apparently, the phase separation occurs mainly in the droplets, hence has no or at least minimal influence 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 (b) 5µm 1µm 1µm (c) 1µm 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. Copyright # 5 John Wiley & Sons, Ltd. Polym. Adv. Technol. 5; 16:

4 Preparation of porous polylactide microspheres 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 68C) has yielded porous microspheres with same surface morphology. 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 polymerlean 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 microspheres. 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 rate Keeping other conditions constant, the influence of stirring rate was firstly investigated with respect to particle size, par- (a) 625 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 to 8 rpm, the number average diameter of the particles was decreased from 47 to 18 mm, respectively (Table 1). All the particle size distributions were quite narrow and accordingly shifted to the smaller 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 Theoretically, 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,.61. To testify this influence and to compare the apparent density of porous particles with nonporous ones, non-porous microspheres with diameters ranging 1 154, , and mm were similarly characterized, resulting in apparent densities of 1.124, 1.113, 1.83 and 1.49 g/cm3, respectively. These data indeed demonstrated that the apparent density of the nonporous spheres was much bigger than that of the porous ones, (b) 5µm (c) 5µm 5µm (d) 5µm 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. Copyright # 5 John Wiley & Sons, Ltd. Polym. Adv. Technol. 5; 16:

5 626 Y. Hong et al. 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.61. 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 ratio Good solvent/non-solvent ratio is another key factor controlling 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 produced. As expected, when the ratio of the good solvent/poor solvent decreased from 9.5:.5 to 8:2, the pore size increased from 5 to 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 emulsionsolvent 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 concentration The 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 (a) (b) µm µm (c) (d) µm µm Figure 3. Particle size distribution histograms of the porous microspheres. (a) Sample A, (b) sample B, (c) sample C, and (d) sample E. Copyright # 5 John Wiley & Sons, Ltd. Polym. Adv. Technol. 5; 16:

6 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 concentration has shown a reverse effect with the good solvent/nonsolvent ratio. As shown in Table 1, the pore size decreased from 4to7 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 concentration The 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 apparent density and pore size. It is well understood that a higher concentration of dispersant is beneficial to decrease the interfacial energy of oil droplets, thus smaller droplets that correspond to larger surface area can be formed. As discussed earlier, the apparent density increase would not mean a porosity decrease in the present case. With higher PVA concentration, 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 successfully 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. to.45 g/cm 3, was much smaller than that of the non-porous microspheres (>1 g/cm 3 ). 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 18 to 47 mm. Moreover, these fabrication factors can also influence the apparent density more or less either by size variance or porosity alteration or both. Acknowledgements This study is financially supported by the Natural Science Foundation of China (Nos 434 and 966) and the National Science Fund for Distinguished Young Scholars of China (No ). REFERENCES 1. Van der Elst M, Klein CPAT, de Blieck-Hogervorst JM, Patka P, Haarman HJThM. Bone tissue response to biodegradable polymers used for intra medullary fracture fixation: a long-term in vivo study in sheep femora. Biomaterials 1999; : Bergma J, de Bruijin WD, Rozema FR, Bos RRM, Boering G. 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