Journal of Colloid and Interface Science 465 (2016) 18–25
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Preparation of nano/macroporous polycaprolactone microspheres for an injectable cell delivery system using room temperature ionic liquid and camphene Seong Yeol Kim a,b, Ji-Young Hwang c,⇑, Ueon Sang Shin a,b,⇑ a Department of Nanobiomedical Science & BK21 PlUS NBM Global Research Center for Regenerative Medicine, Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea b Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea c Department of Biomedical Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-713, Republic of Korea
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 12 October 2015 Revised 20 November 2015 Accepted 20 November 2015 Available online 22 November 2015 Keywords: Porous PCL microspheres Nano- and micro-porous surface morphology An injectable cell delivery system Room temperature ionic liquids NGF-loaded microspheres
a b s t r a c t The nano/macroporous polycaprolactone (PCL) microspheres with cell active surfaces were developed as an injectable cell delivery system. Room temperature ionic liquid (RTIL) and camphene were used as a liquid mold and a porogen, respectively. Various-sized spheres of 244–601 lm with pores of various size and shape of 0.02–100 lm, were formed depending on the camphene/RTIL ratio (0.8–2.6). To give cell activity, the surface of porous microspheres were further modified with nerve growth factors (NGF) containing gelatin to give a thin NGF/gelatin layer, to which the neural progenitor cells (PC-12) attached and extended their neurites on to the surface layers of the microspheres. The developed microspheres may be potentially applicable as a neuronal cell delivery scaffold for neuron tissue engineering. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors at: Department of Nanobiomedical Science & BK21 PlUS NBM Global Research Center for Regenerative Medicine, Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea (U.S. Shin). E-mail address: [email protected] (U.S. Shin). http://dx.doi.org/10.1016/j.jcis.2015.11.055 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction In the field of tissue engineering, development of porous microspheres has been intensively attempted as an injectable cell
S.Y. Kim et al. / Journal of Colloid and Interface Science 465 (2016) 18–25
delivery system. Cells could be attached, proliferated, onto the porous microspheres, and implanted into a damaged site to induce new-tissue formation [1–7]. Compared to the smooth nonporous surface of the microsphere system, the porous nature can provide a larger specific surface area and more efficient cell expansion [5,8,9]. It is therefore critical to create pore-structured microspheres. Porous microspheres of biopolymers have been fabricated conventionally by a variety of techniques including: emulsion/ solvent evaporation (phase separation) and forming gas pockets [7,10–12]; solution spraying method [13]; blended polymer extraction [14]; spinning disk atomization [1]; and self-assembly [8]. The phase separation technique is the most popular due to the possibility of large-scale production, uniformity of the sphere size, and simple process without the usage of complicated instruments. Due to biocompatible and slow biodegradable characteristics, PCL has generally been used to prepare porous microspheres to support and deliver cells [15–19]. To address many of the difficulties associated with traditional methods of administration, microspheres with a diameter in the range of 300–500 lm are most desirable and as such are being actively developed as a cell delivery system. Ionic liquids (ILs) consist of bulky and asymmetric organic cations such as 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, N-methyl-N-alkylpyrrolidinium and ammonium ions. They can bear a wide range of counter anions, for example, simple halides such as Cl and Br , to large inorganic and organic anions such as tetrafluoroborate (BF4 ), hexafluoroantimonate (SbF6 ), hexafluorophosphate (PF6 ), bistriflimide (NTf2 ) and triflate (OTf ). ILs with a melting point below 100 °C have become known as the room temperature ionic liquids (RTILs), and typical examples include: 1-butyl-3-methylimidazolium (bmim) or ammonium based ionic liquids bearing such anions as SbF6 , PF6 , NTf2 , OTf , BF4 , or Cl . Some of them are in liquid form even at much lower temperatures, such as [bmim]BF4 which is liquid at temperatures of > 80 °C and trioctylmethylammonium chloride (TOMAC) which is liquid at temperatures of > 20 °C. RTILs have many fascinating properties, for example, these liquids have strong polar and nonpolar parts and they can be miscible and/or immiscible with water and a number of organic solvents [20–22]. This is the case since, the types of organic cations and inorganic anions of RTILs greatly affect their wettability, i.e., hydrophilic/hydrophobic nature. The constituents of RTILs are constrained by high coulombic forces, exhibiting practically no vapor pressure even at an elevated temperature. These unique traits of RTILs allow the possibility for diverse use throughout basic science and industry [23,24]. In our previous research to prepare non-porous polymeric microspheres and porous membranes [25–28], we fabricated drug-loaded porous microspheres of PCL using an ammonium-based RTIL as a liquid mold for microsphere formation and camphene as a porogen for pore formation. The porous PCL microspheres, which are soccer ball-like in appearance, have a size of 244–601 lm in diameter, and various pore sizes of 20–2000 nm for wave-type of pores on the surface and various surface holes of 10–100 lm interconnected to internal pores. Utilization of various combinations of RTIL and camphene for tuning sphere and pore-size is reported for the first time herein and will open up a new way to produce polymeric carriers for use, not only as drugs but also in cells.
19
purchased from Korean Sigma Aldrich, respectively, as an RTIL, an organic solvent, and a porogen. Nerve growth factor (NGF), which was purchased from Korean Sigma Aldrich, was secreted as a model protein that is important for the growth, maintenance, and survival of certain target neurons. The materials were used without further purification. The 3D morphology of the porous PCL microspheres were examined with scanning electron microscopy (SEM) (JEOL and HITACHI S-3000H, Japan) and the physicochemical properties were examined using an FT-IR spectrometer (Perkin-Elmer Spectrum BXII, US). For SEM analysis, samples were sputter-coated with approximately 10 nm of gold, before analysis. The IR spectra were recorded between 4000 and 500 cm 1, with 16 scans at 4 cm 1 resolution. The purity of product was examined by thermogravimetric analysis (TGA), which was conducted using a Seiko Exstar 6000 TG/ DTA6100 (SEICO INST., JAPAN) with a heating rate of 10 °C/min at temperatures ranging from 20 to 1000 °C. 2.2. Fabrication of porous PCL microspheres The porous PCL microspheres were prepared as follows: 0.1 g of PCL, 2.5 g of the RTIL, and 2 g of camphene were dissolved in 50 mL of DCM to produce a transparent solution. The organic solvent (DCM) was then evaporated from the transparent solution under ambient conditions. Rubbery white gels remained at the bottom of the glass dish. To remove the RTIL, the rubbery cake obtained was dissolved in ethanol and the porous micro particles were collected by centrifugation at 3000 rpm for 10 min. This washing process was repeated 3 times and the obtained white microspheres were dried under ambient conditions. The ionic liquid recovered was reused for the next run. 2.3. NGF/gelatin-coating on the surface of PCL microspheres To prepare NGF/gelatin mixture-coated PCTpmb-2 (NGF/Gel/ PCLpms-2) microspheres, the following steps were taken: 50 mg of porous microspheres were placed in a flask, following vacuumization. 5 ml of gelatin solution (0.1 wt%) containing 5 lg NGF was injected into the flask under continuous shaking. After removing any gelatin residues from the PCL microsphere surface with water, the PCL microspheres containing gelatin-NGF mixture in their various pores were freeze-dried. 2.4. In vitro cell culture A rat adrenal pheochromocytoma-derived cell line, PC-12, (American Type Culture Collection, Manassas, VA) was used to test cell behavior of NGF/gelatin mixture-coated PCTpmb-2 (NGF/Gel/ PCLpms-2) microspheres. It was found that it reversibly responded to NGF by induction of the neuronal phenotype, ceasing cell division and extending neurites. PC-12 cells were cultivated in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD) containing 10% (v/v) fetal bovine serum (Gibco), 100 units/mL penicillin and 100 g/mL streptomycin (Gibco) at 37 °C in a 5% CO2 atmosphere, in a humidified incubator (Thermo Scientific Inc., Waltham, MA). 2.5. Preparation of each microsphere group for neuronal cell behaviors
2. Experimental 2.1. Materials and characterization Polycaprolactone (PCL) as a biodegradable polymer was purchased from Boehringer Ingelheim. Trioctylmethylammonium chloride (TOMAC), CH2Cl2 (DCM), and camphene which were
For the cell behavior study, effects of NGF on cell viability, cell morphology and neuronal differentiation of PC-12 cells were examined on PCLpms-2, Gel/PCLpms-2 and PCNGpms-2 (NGF/ Gel/PCLpms-2) microspheres. Each microsphere group was first immersed in PBS in a 1.5 ml Eppendorf tube, left for several hours until the microspheres had sunk to the bottom of the tube, and
20
S.Y. Kim et al. / Journal of Colloid and Interface Science 465 (2016) 18–25
then were washed with culture medium three times, before sterilization by ultraviolet (UV) irradiation for 30 min before use. In this study, we chose to use 12 mm polycarbonate membrane cell culture inserts with 3.0 lm pore size (Millipore, Billerica, MA) to hold both the microspheres and the cells. The inserts were placed into a 24-well cell-culture plate and allowed to soak in culture medium for several minutes, after which a 1.0 mg aliquot of each microsphere was transferred into an insert. The PC-12 cells were seeded on to each microsphere in the cell culture inserts, at a cell density of 100,000 cells/well, and were then cultivated for five days. 2.6. Analysis of cell morphology Microspheres with the neuronal cells were fixed with 2.5% glutaraldehyde, dehydrated through a series of ethanol concentrations (50%, 75%, 95%, and 100%), and then treated with hexamethyldisilazane, followed by drying of the samples in the hood overnight. The surface morphology of the PC-12 cells on the microspheres was observed by FE-SEM (MIRA II, Tescan, Czech Republic) operated at an accelerating voltage of 1.0 kV using standard procedures. 2.7. Statistics All experiments were repeated at least three times, and the data presented are means ± standard deviation (S.D.). Statistical analysis was performed using the SPSS 12.0 software (SPSS, Chicago, IL). Statistical significance between groups was evaluated with a two-tailed Student’s t-test. Differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Control of sphere size by changing the ratio of camphene/TOMAC We attempted to develop an injectable cell delivery system with a porous spherical shape using PCL solution in DCM containing TOMAC as a liquid mold, and camphene as a porogen. Upon evaporation of the organic solvent from the solution at room temperature, a transition from a homogeneous liquid state to a gel state was observed. Selective extraction of the RTIL phase, which was surrounding the camphene-enclosed or -embedded PCL microspheres, from the polymer gel with 100% ethanol, yielded white soccer ball-like, porous microspheres (Fig. 1b–d). Compared with non-porous PCL microspheres, PCLms, as shown in Fig. 1a [25], which were prepared without campene, the porous PCL microspheres (PCLpms-13), which are all football-like in appearance, showed a size of 244–601 lm in sphere diameter (Fig. 1b–d and Table 1). The sphere size control was achieved by applying various combinations of the RTIL and camphene. When the concentration ratio of camphene/TOMAC (the TOMAC concentration is constant) increase from 0.8 to 1.6 and 2.8, the sphere-sizes increased gradually from 244 to 390 and 601 lm in diameter. Compared to PCLms, the mean sizes of PCLpms-13 grew up to 70–176 times. 3.2. Dependance of pore-size, -shape, and -number on the ratio of camphene/TOMAC The weight ratio of camphene/TOMAC greatly influenced the pore morphology and number, as summarized in Table 1. When the ratio was 0 (no campene used), no open pores were observed on the sphere surface (PCLms) as shown in Fig. 2a. However, when the ratio was increased to 0.8, open pores of 10–20 lm diameter were observed on the sphere surface (PCLpms-1) as shown in
Fig. 2b. A further increase in the ratio of camphene/TOMAC to 1.6 and 2.4 for the samples of PCLpms-2 and -3, respectively, led to the further increase of pore-size (20–40 and 40–100 lm; approximately two to five times, respectively) and pore-number (approximately three to four times, respectively). To see the internal structure of porous PCL microspheres (using PCLpms-2), the samples were frozen in liquid nitrogen and broken up into fine particles. The internal morphology of the broken particles also appeared to be highly porous and to have interconnected pore channels (Fig. 3). Consequently, it was considered that their regular and highly porous phase and larger pore size may have resulted from a partially higher camphene concentration [29,30]. Moreover, it is notable that only a low weight ratio, not exceeding 2.5, was required to create highly porous external morphology and interconnective internal pore channels. 3.3. The surface nano-morphology of microspheres changes with the ratio of camphene/TOMAC The weight ratio of camphene/TOMAC also influenced the surface nano-morphology of microspheres, for instance, formation of wave-like patterns of nanopores in sizes of 20–2000 nm (see Table 1). The average pore size of a wave pore could be actively controlled by altering the weight ratio of camphene/TOMAC (or the camphene concentration). As shown in Fig. 4, the surface of PCLms prepared without camphene, showed only flat and smooth topography without any nanopores, whereas the surface of all the PCLpms-1 to -3 microspheres prepared in the presence of various combinations of camphene/TOMAC, were covered with wave like pattern of nanopores beside the hole-type of micro-sized pores. In the case of PCLpms-1, a weak, rough surface was found to have an irregular wave-like pores of about 20–30 nm wide. However, the surfaces of PCLpms-2 and -3 microspheres were covered with more, regularly spaced wave-like pores of about 200–2000 nm wide. These surface nano-patterns became gradually stronger because of the increased concentration of camphene in the PCL/ TOMAC precursor solution. Camphene molecules initially penetrated into the inside of the microspheres and the remainder surrounded the outside during the solvent-drying process. When increasing the concentration of the surplus camphene, it could be that more camphene regularly accumulated on the surface of globular PCL microbeads, and then the wave-like pore structures became larger and regular. 3.4. Mechanistic demonstration for the formation of PCLpms in the presence of camphene/TOMAC The proposed mechanism for the formation of the soccer ball-like porous PCL microspheres (PCLpms) is schematically represented in Fig. 5. PCL and TOMAC were dissolved in homogenous DCM solution containing camphene, which led to the formation of a viscous solution. Upon evaporation of the solvent at room temperature, a state transition from a homogeneous fluid to a gel (or a phase separation into hydrophobic PCL/camphene phase and hydrophilic TOMAC phase in micro scale) took place [25]. We propose that, in the first step, a portion of the introduced camphene could move into the spherical PCL phase to form small-sized PCL microspheres (PCLms complexes of about 3–5 lm in diameter) embedding camphene molecules [25]. The remaining camphene could be deposited on the surface of the PCL microspheres together with TOMAC molecules, because camphene molecules easily aggregate due to their hydrophobicity. Upon the continued evaporation of the solvent, the in situ-formed non-porous PCLms complexes group together with the hydrophobic camphene and TOMAC components. These might be closely conglomerated together by strong interactions between their hydrophobic surfaces (mainly through
S.Y. Kim et al. / Journal of Colloid and Interface Science 465 (2016) 18–25
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Fig. 1. SEMs showing increasing size of porous PCL microspheres: (a) PCLms; (b) PCLpms-1; (c) PCLpms-2; (d) PCLpms-3.
Table 1 Relationship of the ratio of camphene/TOMAC with mean sphere size, mean pore size, and pore shape. Sample name
PCLms PCLpms-1 PCLpms-2 PCLpms-3
Camphene/ Sphere TOMAC (g) diameter (lm)
0/2.5 2.0/2.5 (=0.8) 4.0/2.5 (=1.6) 6.0/2.5 (=2.4)
3.41 ± 1.07 244 ± 45.51 390 ± 71.51 601 ± 97.23
Surface morphology
Wave-like pore (nm)
Round-shaped pore (lm)
Flat and smooth Pores between waves: 20–30 Pores between waves: 200–300 Pores between waves: 200–2000
No pore 10–20 20–40 40–100
the hydrophobic characteristic of PCL/camphene) of the nonporous PCLms complexes to form larger and soccer ball-like PCL microspheres (as an aggregate of the PCLms complexes). When the solvent has completely evaporated, camphene component may also be volatile and make internal and external surface micropores containing the wave like pattern of nano-pores, resulting in the formation of the soccer ball-like porous PCL microspheres (PCLpms).
3.5. NGF/gelatin mixture-coated PCNGpms-2 (NGF/Gel/PCLpms-2) microspheres For efficient cell attachment and proliferation on the porous PCL microspheres and for effective implantation of the porous PCL microsphere-cell complexes into damaged sites to induce newtissue formation, the PCLpms-2 microspheres were selected as a model cell-carrier among the three samples (PCLpms-13). Their
surfaces were coated with NGF-containing gelatin solution (0.1 wt% gelatin, 0.0001 wt% NGF) to generate NGF/gelatin mixture-coated PCNGpms-2 (NGF/Gel/PCLpms-2) microspheres. as shown in the SEM micrographs in Fig. 6, the number and size of the surface pores appeared significantly reduced. After coating with gelatin/NGF mixture the pores with the original size of about 20–40 lm in diameter, were changed to small pores of
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Preparation of nano/macroporous polycaprolactone microspheres for an injectable cell delivery system using room temperature ionic liquid and camphene Seong Yeol Kim a,b, Ji-Young Hwang c,⇑, Ueon Sang Shin a,b,⇑ a Department of Nanobiomedical Science & BK21 PlUS NBM Global Research Center for Regenerative Medicine, Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea b Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea c Department of Biomedical Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-713, Republic of Korea
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 12 October 2015 Revised 20 November 2015 Accepted 20 November 2015 Available online 22 November 2015 Keywords: Porous PCL microspheres Nano- and micro-porous surface morphology An injectable cell delivery system Room temperature ionic liquids NGF-loaded microspheres
a b s t r a c t The nano/macroporous polycaprolactone (PCL) microspheres with cell active surfaces were developed as an injectable cell delivery system. Room temperature ionic liquid (RTIL) and camphene were used as a liquid mold and a porogen, respectively. Various-sized spheres of 244–601 lm with pores of various size and shape of 0.02–100 lm, were formed depending on the camphene/RTIL ratio (0.8–2.6). To give cell activity, the surface of porous microspheres were further modified with nerve growth factors (NGF) containing gelatin to give a thin NGF/gelatin layer, to which the neural progenitor cells (PC-12) attached and extended their neurites on to the surface layers of the microspheres. The developed microspheres may be potentially applicable as a neuronal cell delivery scaffold for neuron tissue engineering. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding authors at: Department of Nanobiomedical Science & BK21 PlUS NBM Global Research Center for Regenerative Medicine, Dankook University, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea (U.S. Shin). E-mail address: [email protected] (U.S. Shin). http://dx.doi.org/10.1016/j.jcis.2015.11.055 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction In the field of tissue engineering, development of porous microspheres has been intensively attempted as an injectable cell
S.Y. Kim et al. / Journal of Colloid and Interface Science 465 (2016) 18–25
delivery system. Cells could be attached, proliferated, onto the porous microspheres, and implanted into a damaged site to induce new-tissue formation [1–7]. Compared to the smooth nonporous surface of the microsphere system, the porous nature can provide a larger specific surface area and more efficient cell expansion [5,8,9]. It is therefore critical to create pore-structured microspheres. Porous microspheres of biopolymers have been fabricated conventionally by a variety of techniques including: emulsion/ solvent evaporation (phase separation) and forming gas pockets [7,10–12]; solution spraying method [13]; blended polymer extraction [14]; spinning disk atomization [1]; and self-assembly [8]. The phase separation technique is the most popular due to the possibility of large-scale production, uniformity of the sphere size, and simple process without the usage of complicated instruments. Due to biocompatible and slow biodegradable characteristics, PCL has generally been used to prepare porous microspheres to support and deliver cells [15–19]. To address many of the difficulties associated with traditional methods of administration, microspheres with a diameter in the range of 300–500 lm are most desirable and as such are being actively developed as a cell delivery system. Ionic liquids (ILs) consist of bulky and asymmetric organic cations such as 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, N-methyl-N-alkylpyrrolidinium and ammonium ions. They can bear a wide range of counter anions, for example, simple halides such as Cl and Br , to large inorganic and organic anions such as tetrafluoroborate (BF4 ), hexafluoroantimonate (SbF6 ), hexafluorophosphate (PF6 ), bistriflimide (NTf2 ) and triflate (OTf ). ILs with a melting point below 100 °C have become known as the room temperature ionic liquids (RTILs), and typical examples include: 1-butyl-3-methylimidazolium (bmim) or ammonium based ionic liquids bearing such anions as SbF6 , PF6 , NTf2 , OTf , BF4 , or Cl . Some of them are in liquid form even at much lower temperatures, such as [bmim]BF4 which is liquid at temperatures of > 80 °C and trioctylmethylammonium chloride (TOMAC) which is liquid at temperatures of > 20 °C. RTILs have many fascinating properties, for example, these liquids have strong polar and nonpolar parts and they can be miscible and/or immiscible with water and a number of organic solvents [20–22]. This is the case since, the types of organic cations and inorganic anions of RTILs greatly affect their wettability, i.e., hydrophilic/hydrophobic nature. The constituents of RTILs are constrained by high coulombic forces, exhibiting practically no vapor pressure even at an elevated temperature. These unique traits of RTILs allow the possibility for diverse use throughout basic science and industry [23,24]. In our previous research to prepare non-porous polymeric microspheres and porous membranes [25–28], we fabricated drug-loaded porous microspheres of PCL using an ammonium-based RTIL as a liquid mold for microsphere formation and camphene as a porogen for pore formation. The porous PCL microspheres, which are soccer ball-like in appearance, have a size of 244–601 lm in diameter, and various pore sizes of 20–2000 nm for wave-type of pores on the surface and various surface holes of 10–100 lm interconnected to internal pores. Utilization of various combinations of RTIL and camphene for tuning sphere and pore-size is reported for the first time herein and will open up a new way to produce polymeric carriers for use, not only as drugs but also in cells.
19
purchased from Korean Sigma Aldrich, respectively, as an RTIL, an organic solvent, and a porogen. Nerve growth factor (NGF), which was purchased from Korean Sigma Aldrich, was secreted as a model protein that is important for the growth, maintenance, and survival of certain target neurons. The materials were used without further purification. The 3D morphology of the porous PCL microspheres were examined with scanning electron microscopy (SEM) (JEOL and HITACHI S-3000H, Japan) and the physicochemical properties were examined using an FT-IR spectrometer (Perkin-Elmer Spectrum BXII, US). For SEM analysis, samples were sputter-coated with approximately 10 nm of gold, before analysis. The IR spectra were recorded between 4000 and 500 cm 1, with 16 scans at 4 cm 1 resolution. The purity of product was examined by thermogravimetric analysis (TGA), which was conducted using a Seiko Exstar 6000 TG/ DTA6100 (SEICO INST., JAPAN) with a heating rate of 10 °C/min at temperatures ranging from 20 to 1000 °C. 2.2. Fabrication of porous PCL microspheres The porous PCL microspheres were prepared as follows: 0.1 g of PCL, 2.5 g of the RTIL, and 2 g of camphene were dissolved in 50 mL of DCM to produce a transparent solution. The organic solvent (DCM) was then evaporated from the transparent solution under ambient conditions. Rubbery white gels remained at the bottom of the glass dish. To remove the RTIL, the rubbery cake obtained was dissolved in ethanol and the porous micro particles were collected by centrifugation at 3000 rpm for 10 min. This washing process was repeated 3 times and the obtained white microspheres were dried under ambient conditions. The ionic liquid recovered was reused for the next run. 2.3. NGF/gelatin-coating on the surface of PCL microspheres To prepare NGF/gelatin mixture-coated PCTpmb-2 (NGF/Gel/ PCLpms-2) microspheres, the following steps were taken: 50 mg of porous microspheres were placed in a flask, following vacuumization. 5 ml of gelatin solution (0.1 wt%) containing 5 lg NGF was injected into the flask under continuous shaking. After removing any gelatin residues from the PCL microsphere surface with water, the PCL microspheres containing gelatin-NGF mixture in their various pores were freeze-dried. 2.4. In vitro cell culture A rat adrenal pheochromocytoma-derived cell line, PC-12, (American Type Culture Collection, Manassas, VA) was used to test cell behavior of NGF/gelatin mixture-coated PCTpmb-2 (NGF/Gel/ PCLpms-2) microspheres. It was found that it reversibly responded to NGF by induction of the neuronal phenotype, ceasing cell division and extending neurites. PC-12 cells were cultivated in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD) containing 10% (v/v) fetal bovine serum (Gibco), 100 units/mL penicillin and 100 g/mL streptomycin (Gibco) at 37 °C in a 5% CO2 atmosphere, in a humidified incubator (Thermo Scientific Inc., Waltham, MA). 2.5. Preparation of each microsphere group for neuronal cell behaviors
2. Experimental 2.1. Materials and characterization Polycaprolactone (PCL) as a biodegradable polymer was purchased from Boehringer Ingelheim. Trioctylmethylammonium chloride (TOMAC), CH2Cl2 (DCM), and camphene which were
For the cell behavior study, effects of NGF on cell viability, cell morphology and neuronal differentiation of PC-12 cells were examined on PCLpms-2, Gel/PCLpms-2 and PCNGpms-2 (NGF/ Gel/PCLpms-2) microspheres. Each microsphere group was first immersed in PBS in a 1.5 ml Eppendorf tube, left for several hours until the microspheres had sunk to the bottom of the tube, and
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S.Y. Kim et al. / Journal of Colloid and Interface Science 465 (2016) 18–25
then were washed with culture medium three times, before sterilization by ultraviolet (UV) irradiation for 30 min before use. In this study, we chose to use 12 mm polycarbonate membrane cell culture inserts with 3.0 lm pore size (Millipore, Billerica, MA) to hold both the microspheres and the cells. The inserts were placed into a 24-well cell-culture plate and allowed to soak in culture medium for several minutes, after which a 1.0 mg aliquot of each microsphere was transferred into an insert. The PC-12 cells were seeded on to each microsphere in the cell culture inserts, at a cell density of 100,000 cells/well, and were then cultivated for five days. 2.6. Analysis of cell morphology Microspheres with the neuronal cells were fixed with 2.5% glutaraldehyde, dehydrated through a series of ethanol concentrations (50%, 75%, 95%, and 100%), and then treated with hexamethyldisilazane, followed by drying of the samples in the hood overnight. The surface morphology of the PC-12 cells on the microspheres was observed by FE-SEM (MIRA II, Tescan, Czech Republic) operated at an accelerating voltage of 1.0 kV using standard procedures. 2.7. Statistics All experiments were repeated at least three times, and the data presented are means ± standard deviation (S.D.). Statistical analysis was performed using the SPSS 12.0 software (SPSS, Chicago, IL). Statistical significance between groups was evaluated with a two-tailed Student’s t-test. Differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Control of sphere size by changing the ratio of camphene/TOMAC We attempted to develop an injectable cell delivery system with a porous spherical shape using PCL solution in DCM containing TOMAC as a liquid mold, and camphene as a porogen. Upon evaporation of the organic solvent from the solution at room temperature, a transition from a homogeneous liquid state to a gel state was observed. Selective extraction of the RTIL phase, which was surrounding the camphene-enclosed or -embedded PCL microspheres, from the polymer gel with 100% ethanol, yielded white soccer ball-like, porous microspheres (Fig. 1b–d). Compared with non-porous PCL microspheres, PCLms, as shown in Fig. 1a [25], which were prepared without campene, the porous PCL microspheres (PCLpms-13), which are all football-like in appearance, showed a size of 244–601 lm in sphere diameter (Fig. 1b–d and Table 1). The sphere size control was achieved by applying various combinations of the RTIL and camphene. When the concentration ratio of camphene/TOMAC (the TOMAC concentration is constant) increase from 0.8 to 1.6 and 2.8, the sphere-sizes increased gradually from 244 to 390 and 601 lm in diameter. Compared to PCLms, the mean sizes of PCLpms-13 grew up to 70–176 times. 3.2. Dependance of pore-size, -shape, and -number on the ratio of camphene/TOMAC The weight ratio of camphene/TOMAC greatly influenced the pore morphology and number, as summarized in Table 1. When the ratio was 0 (no campene used), no open pores were observed on the sphere surface (PCLms) as shown in Fig. 2a. However, when the ratio was increased to 0.8, open pores of 10–20 lm diameter were observed on the sphere surface (PCLpms-1) as shown in
Fig. 2b. A further increase in the ratio of camphene/TOMAC to 1.6 and 2.4 for the samples of PCLpms-2 and -3, respectively, led to the further increase of pore-size (20–40 and 40–100 lm; approximately two to five times, respectively) and pore-number (approximately three to four times, respectively). To see the internal structure of porous PCL microspheres (using PCLpms-2), the samples were frozen in liquid nitrogen and broken up into fine particles. The internal morphology of the broken particles also appeared to be highly porous and to have interconnected pore channels (Fig. 3). Consequently, it was considered that their regular and highly porous phase and larger pore size may have resulted from a partially higher camphene concentration [29,30]. Moreover, it is notable that only a low weight ratio, not exceeding 2.5, was required to create highly porous external morphology and interconnective internal pore channels. 3.3. The surface nano-morphology of microspheres changes with the ratio of camphene/TOMAC The weight ratio of camphene/TOMAC also influenced the surface nano-morphology of microspheres, for instance, formation of wave-like patterns of nanopores in sizes of 20–2000 nm (see Table 1). The average pore size of a wave pore could be actively controlled by altering the weight ratio of camphene/TOMAC (or the camphene concentration). As shown in Fig. 4, the surface of PCLms prepared without camphene, showed only flat and smooth topography without any nanopores, whereas the surface of all the PCLpms-1 to -3 microspheres prepared in the presence of various combinations of camphene/TOMAC, were covered with wave like pattern of nanopores beside the hole-type of micro-sized pores. In the case of PCLpms-1, a weak, rough surface was found to have an irregular wave-like pores of about 20–30 nm wide. However, the surfaces of PCLpms-2 and -3 microspheres were covered with more, regularly spaced wave-like pores of about 200–2000 nm wide. These surface nano-patterns became gradually stronger because of the increased concentration of camphene in the PCL/ TOMAC precursor solution. Camphene molecules initially penetrated into the inside of the microspheres and the remainder surrounded the outside during the solvent-drying process. When increasing the concentration of the surplus camphene, it could be that more camphene regularly accumulated on the surface of globular PCL microbeads, and then the wave-like pore structures became larger and regular. 3.4. Mechanistic demonstration for the formation of PCLpms in the presence of camphene/TOMAC The proposed mechanism for the formation of the soccer ball-like porous PCL microspheres (PCLpms) is schematically represented in Fig. 5. PCL and TOMAC were dissolved in homogenous DCM solution containing camphene, which led to the formation of a viscous solution. Upon evaporation of the solvent at room temperature, a state transition from a homogeneous fluid to a gel (or a phase separation into hydrophobic PCL/camphene phase and hydrophilic TOMAC phase in micro scale) took place [25]. We propose that, in the first step, a portion of the introduced camphene could move into the spherical PCL phase to form small-sized PCL microspheres (PCLms complexes of about 3–5 lm in diameter) embedding camphene molecules [25]. The remaining camphene could be deposited on the surface of the PCL microspheres together with TOMAC molecules, because camphene molecules easily aggregate due to their hydrophobicity. Upon the continued evaporation of the solvent, the in situ-formed non-porous PCLms complexes group together with the hydrophobic camphene and TOMAC components. These might be closely conglomerated together by strong interactions between their hydrophobic surfaces (mainly through
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Fig. 1. SEMs showing increasing size of porous PCL microspheres: (a) PCLms; (b) PCLpms-1; (c) PCLpms-2; (d) PCLpms-3.
Table 1 Relationship of the ratio of camphene/TOMAC with mean sphere size, mean pore size, and pore shape. Sample name
PCLms PCLpms-1 PCLpms-2 PCLpms-3
Camphene/ Sphere TOMAC (g) diameter (lm)
0/2.5 2.0/2.5 (=0.8) 4.0/2.5 (=1.6) 6.0/2.5 (=2.4)
3.41 ± 1.07 244 ± 45.51 390 ± 71.51 601 ± 97.23
Surface morphology
Wave-like pore (nm)
Round-shaped pore (lm)
Flat and smooth Pores between waves: 20–30 Pores between waves: 200–300 Pores between waves: 200–2000
No pore 10–20 20–40 40–100
the hydrophobic characteristic of PCL/camphene) of the nonporous PCLms complexes to form larger and soccer ball-like PCL microspheres (as an aggregate of the PCLms complexes). When the solvent has completely evaporated, camphene component may also be volatile and make internal and external surface micropores containing the wave like pattern of nano-pores, resulting in the formation of the soccer ball-like porous PCL microspheres (PCLpms).
3.5. NGF/gelatin mixture-coated PCNGpms-2 (NGF/Gel/PCLpms-2) microspheres For efficient cell attachment and proliferation on the porous PCL microspheres and for effective implantation of the porous PCL microsphere-cell complexes into damaged sites to induce newtissue formation, the PCLpms-2 microspheres were selected as a model cell-carrier among the three samples (PCLpms-13). Their
surfaces were coated with NGF-containing gelatin solution (0.1 wt% gelatin, 0.0001 wt% NGF) to generate NGF/gelatin mixture-coated PCNGpms-2 (NGF/Gel/PCLpms-2) microspheres. as shown in the SEM micrographs in Fig. 6, the number and size of the surface pores appeared significantly reduced. After coating with gelatin/NGF mixture the pores with the original size of about 20–40 lm in diameter, were changed to small pores of
