Engineering PLGA doped PCL microspheres with a layered architecture and an island–sea topography

Abstract

Composite polymer devices have gained considerable interest in biomedical applications. In this work, a composite poly(lactic-co-glycolic acid)/poly(ε-caprolactone) (PLGA/PCL) microsphere with a layered architecture and an island–sea topography was developed. PCL was found to occupy the shell layer of the microsphere, while PLGA constituted surface islands and the internal core. This subtle structure was derived from the different external and internal rates of phase separation between PCL and PLGA. The existence of the PLGA islands enhanced the hydrophilicity of the PCL shell. Mouse mesenchymal stem cells (mMSCs) were cultured and they grew well on the PLGA/PCL microspheres. The surface of the microsphere was microscopically composed of PCL sea and PLGA islands, and the mMSCs showed a trend to attach to the more hydrophilic PLGA islands and bridged between adjacent microspheres. This paper provides a new alternative to design functional microspheres for potential application in tissue repair.

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1. Introduction

Biopolymers play an increasingly important role in tissue repair.^1,2 They degrade in vivo and thus no extra effort is required to extract them out of the body.³ Biopolymer devices can act as drug carriers and prolong or target the release of various therapeutic agents.^4,5 Moreover, biopolymers can be designed into diverse scaffolds, which then incorporate with specific cells to repair tissues.^6,7 Since a single polymer has limitations in one or more aspects such as the mechanical strength, drug delivery or cell affinity, a blend composite is considered a good choice to obtain devices with comprehensive functions.⁸

Poly(ε-caprolactone) (PCL) is a well-known biodegradable and biocompatible polymer.⁹ It has been used as a suture and a drug vehicle for decades. Nowadays, PCL is increasingly studied with respect to bone repair.^10,11 However, one inherent problem with PCL is that it has an unfavorable hydrophobicity characteristic. Many studies have tried to address this issue via doping PCL with poly(lactic-co-glycolic) acid (PLGA). For instance, Tang et al. reported that doping PCL with PLGA improved the hydrophilicity and facilitated the growth of osteoblasts.¹² PLGA doped PCL also has a superior osteoconductivity compared with pure PCL.¹³ So far, the majority of doping attempts are performed on planar films prepared by a casting technique. The surface of the film can directly reflect the composition and morphology features of both the polymers. Nevertheless, planar substrates are hardly applicable as scaffolds in bone repair. Moreover, planar substrates are not usually considered as an ideal drug carrier.

Polymer microspheres have a long history of use as drug vehicles.^14,15 They can also be built into 3-D scaffolds for tissue repair.¹⁶ Composite microspheres with a layered architecture are preferred for drug delivery applications due to their easy and flexible release control.^17,18 PCL, which degrades slower than most other biopolymers, is an ideal candidate for the shell in a layered microsphere. So far, layered microspheres are mainly prepared by blending two immiscible polymers, with one polymer occupying the shell while the other occupying the core.^19,20 Therefore, the surface features only reflect the shell polymer. Accordingly, the doping effect cannot be directly reflected in the shell of a microsphere compared to on planar substrates.

The aim of this study was to fabricate a composite PLGA doped PCL microsphere with a layered architecture and island–sea topography. This subtly designed microsphere was expected to achieve doping on the surface of the layered microsphere. The composition configuration after separation in the microsphere was identified. The formation mechanism was based on the phase separation between PCL and PLGA. Mouse mesenchymal stem cells were cultured on microspheres to explore the effects of the island–sea topography on the growth and adhesion of the cells.

2. Experimental

2.1 Materials

Poly(ε-caprolactone) (PCL, M_w = 80 kDa) and poly(lactic-co-glycolic acid) (PLGA, lactide/glycolide ratio = 50/50, M_w = 31 kDa) were purchased from Daigang Biomaterials (Shandong, China). Poly(vinyl alcohol) (PVA, 87–89% hydrolyzed, M_w = 88 kDa) was obtained from Aladdin Chemistry (Shanghai, China). Dichloromethane and acetone were bought from the Chemical Reagent Factory (Guangzhou, China).

2.2 Preparation of the PLGA/PCL microspheres

PLGA/PCL microspheres were prepared via a single emulsion solvent evaporation technique. PCL and PLGA (1/1, w/w) were dissolved in 5 ml of dichloromethane to obtain the oil phase. The oil phase was then injected into 250 ml of 0.3% (w/v) PVA aqueous solution and emulsified by stirring to form the oil/water emulsion. Dichloromethane (2 ml) was added into the aqueous solution every 10 min three times to facilitate the phase separation between PCL and PLGA. The stirring was continued at 300 rpm for 8 h in a fume hood to remove the organic solvent. The solidified microspheres were collected, washed with deionized water and lyophilized.

2.3 Characterization of the PLGA/PCL microspheres

The morphology of the microspheres was characterized using Quanta 200 SEM (FEI, Netherlands). Samples were mounted on a metal stub and sputter-coated with gold using EM SCD 500 (LEICA, Germany). To observe the internal structure, the microspheres were first cross-sectioned using a scalpel. To identify the component configuration in the microspheres, the intact and cross-sectioned samples were preliminarily treated with acetone before SEM observation.

Raman analysis with a LabRAM Aramis spectrometer (HORIBA Jobin Yvon, France) was utilized to further clarify the configuration of PCL and PLGA in the microspheres. Analysis was performed on the surfaces of a PCL film, a PLGA film and a PLGA/PCL blend film made from the dissolution of the PLGA/PCL microspheres. The components at different areas of the cross-sectioned PLGA/PCL microspheres were also analyzed. Samples were placed under the microscope and irradiated with a 632 nm laser. Data were collected within the spectral window from 4000 to 400 cm⁻¹.

The shrinking ratios of PLGA, PCL and PLGA/PCL microspheres were compared. When the oil phase was emulsified in PVA solution, several oil droplets were taken and photographed using a HiroX microscope (HiroX, Japan). The initial and eventual diameters of the droplets were measured. The shrinking ratio was calculated as the ratio of the initial diameter to the eventual diameter (n = 4).

To compare the hydrophilicity of PLGA, PCL and the PLGA/PCL microspheres, the microspheres were first compressed into discs. The contact angles of these discs were then measured through a sessile drop method in ambient air by using an OCA 15 Contact Angle Meter (Dataphysics, Germany). The contact angles of PLGA and PCL films were also recorded (n = 5).

2.4 Degradation of PLGA/PCL

The PLGA islands were expected to degrade faster than the PCL shell. In vitro degradation was conducted to investigate the change in the morphology. Microspheres were incubated in 10 ml of PBS (pH 7.4) and placed in an orbital shaker at 75 rpm and 37 °C. The PBS was refreshed every ten days and the microspheres were collected at pre-determined time points for SEM observation.

2.5 Cell culture on the microspheres

Mouse mesenchymal stem cells (mMSCs) were obtained from ATCC (CRL-12424, USA). The mMSCs were propagated in high glucose Dulbecco's modified Eagle's medium (Gibco, USA) with 10% (v/v) FBS (Gibco, USA). When the cells had grown near to confluence in 75 mm² flasks, they were exposed to trypsin for 1 min, followed by addition of the culture medium. The mMSCs were centrifuged and then re-suspended in the culture medium. The microspheres were put in 48-well plates coated with 1.5% agarose and sterilized in 75% alcohol for 2 h. After that, the microspheres were washed with PBS and pre-wetted in the culture medium for 12 h. When the medium was extracted, 500 μl of the cell suspension (1 × 10⁴ cells per ml) were seeded on the microspheres (5 × 10³ cells per well). The plates were incubated at 37 °C, 5% CO₂ and 95% humidity for 1, 3 and 5 days. The culture media were refreshed every two days.

2.6 Proliferation of the cells on the microspheres

Proliferation of the cells growing on the microspheres was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan). At each time point, the culture medium was extracted and 200 μl of CCK-8 working solution was added to each cell/microsphere construct. After incubation for 2 h at 37 °C, 100 μl of the solution was taken and the OD value at 450 nm was measured using a 3001 microplate reader (Thermo, USA) (n = 5).

2.7 Adhesion of the cells on the microspheres

To view the adhesion of the mMSCs on the microspheres, the cell/microsphere constructs were washed with PBS and fixed with 2.5% glutaraldehyde for 12 h at 4 °C. The constructs were then dehydrated through a series of graded ethanol (50%, 70%, 80%, 90%, 95% and 100%) for 10 min each. The adhesion of the cells on the microspheres was observed by SEM.

2.8 Statistical analysis

Quantitative experimental results are expressed as mean ± standard deviation. A statistical comparison between two means was determined by the t-test. A value of p < 0.05 was considered to be statistically significant.

3. Results

3.1 Morphology, architecture and composition configuration of the PLGA/PCL microspheres

Fig. 1 shows the morphology and architecture of the PLGA/PCL microspheres. Interestingly, the surface was decorated with numerous regular spherical bumps, generating an island–sea topography (Fig. 1a). These islands were isolated and have definite borders with the sea matrix (Fig. 1b). From the cross-section image, it was found that the microsphere had a definite double-layered architecture (Fig. 1c). Internal cavities were also formed. The islands on the surface were clearly embedded into the sea matrix (Fig. 1d). In the shell, plenty of fine embedded beads could be clearly identified (Fig. 1e). The core was full of fine bumps and several small holes were also observed (Fig. 1f).

Fig. 1 SEM images of the PLGA/PCL microspheres. (a) An entire microsphere with an island–sea topography. (b) A magnified image of the surface. (c) A cross-section of a microsphere showing the layered structure. (d) An image of the islands on a cross-section. (e) A magnified image of the shell. (f) A magnified image of the core.

To figure out the configuration of PCL and PLGA in the microspheres, both intact and cross-sectioned microspheres were treated with acetone. Acetone is a good solvent for dissolving PLGA but does not dissolve PCL readily. The treated intact microsphere (Fig. 2a and b) retained the intact spherical structure, but the surface islands were replaced by concaves, indicating that the islands were dissolved by acetone. This demonstrated that the shell and the islands were composed of PCL and PLGA, respectively. The treated cross-sectioned microsphere had an obvious hollow shell structure (Fig. 2c and d), meaning that the PLGA core had been dissolved. Over a short treatment time, there were many fine beads left, proving that there were PCL components in the PLGA core (Fig. 2c). As the beads dissolved (Fig. 2d), it could be seen that there were also numerous holes present within the shell (Fig. 2e and f), which were derived from the dissolution of the PLGA components. These results proved that the shell consisted of PCL, while the surface islands and the core consisted of PLGA. Moreover, the phase separation was incomplete, with some PLGA remaining in the shell and some PCL remaining in the core.

Fig. 2 Images of PLGA/PCL microspheres after treatment with acetone. (a) An entire microsphere. (b) A magnified image of the surface. (c) A cross-section of a microsphere with beads remaining. The upper right image is the whole cross-section and the bar represents 200 μm. (d) A cross-section of a microsphere after washing the remaining beads. (e) A magnified image of the shell. (f) A magnified image of the inner wall of the shell.

To further verify the phase separation in the microspheres, Raman analysis was conducted and the spectra from the polymer films are shown in Fig. 3a. Characteristic peaks belonging to PCL (2919 cm⁻¹ and 2872 cm⁻¹ (Rectangle I), 1110 cm⁻¹ and 1066 cm⁻¹ (Rectangle II) and 915 cm⁻¹ (Rectangle III)) and characteristic peaks belonging to PLGA (3002 cm⁻¹ and 2945 cm⁻¹ (Rectangle I), 1130 cm⁻¹ (Rectangle II) and 892 cm⁻¹, 874 cm⁻¹ and 848 cm⁻¹ (Rectangle III)) could both be identified in the blend film made from dissolved PLGA/PCL microspheres. This confirmed that the microsphere was composed of PCL and PLGA. In Fig. 3b, characteristic peaks of PCL at 2919 cm⁻¹, 2872 cm⁻¹, 1110 cm⁻¹ and 1066 cm⁻¹ in the shell, and peaks of PLGA at 3002 cm⁻¹, 2945 cm⁻¹ and 1130 cm⁻¹ in the core can be clearly seen. This confirmed that PCL was the main component in the shell while PLGA was the main component in the core. Moreover, a shell peak at 2945 cm⁻¹ assigned to PLGA and a core peak at 2919 cm⁻¹ assigned to PCL could also be observed, implying the incomplete phase separation between PCL and PLGA. These results are in line with the observations in the SEM images.

Fig. 3 Raman spectra of the films and microspheres. (a) PCL film, PLGA film and PLGA/PCL film from the dissolution of PLGA/PCL microspheres; (b) the shell and the core of the cross-sectioned microsphere.

3.2 Formation mechanism of the PLGA/PCL microspheres

PCL and PLGA are essentially immiscible. When the solvent is removed and the polymer concentration rises, PCL and PLGA start phase separating with each other and generate respective rich phases.²¹ According to the spreading coefficient theory,¹⁷ the separating polymers will configure in the most thermodynamically stable way. Three possible ultimate configurations can be obtained: complete engulfing (producing a layered structure), partial engulfing and no engulfing. During the solidification of the PLGA/PCL microspheres, the solvent was removed continuously. PCL and PLGA ultimately separated into a layered architecture based on complete engulfing. However, the continuing solvent evaporation restrained the movement of the PCL and PLGA molecules, leading to an incomplete phase separation. This explained the existence of some PLGA in the shell and some PCL in the core.

With the aim of clarifying the formation mechanism of the island–sea topography, PLGA/PCL films were prepared under different conditions. As the film was prepared via spin-coating at 1000 rpm, small regions of separation (gray circles) were observed (Fig. 4a). When the coating speed was decreased to 200 rpm (Fig. 4b), the separated regions enlarged, generating an island–sea topography similar to that in the PLGA/PCL microspheres. This indicated that a moderate rate of solvent evaporation (i.e. the rate of solidification of the polymer) facilitated the growth of the dispersed phase. If the film was prepared by standing it still in a fume hood (Fig. 4c), the dispersed phase enlarged further, and the area of individual islands became much larger than in the PLGA/PCL microspheres. For the film prepared via standing it still in ambient room conditions (Fig. 4d), the solidification rate was so slow that basically a macroscopic phase separation took place. These results suggested that the generation of the dispersed phase was connected to the solidification rate of the polymer derived from solvent evaporation. A quick solidification allowed limited time for the PLGA molecules to move and assemble, producing dispersed islands instead of a single PLGA-rich phase.

Fig. 4 The SEM images of PLGA/PCL films prepared under different conditions. (a) Spin coating at 1000 rpm; (b) spin coating at 200 rpm; (c) standing still in a fume hood; and (d) standing still in ambient conditions. The rate of solvent evaporation was (a) > (b) > (c) > (d).

If the movement of PLGA molecular chains on the surface of the PLGA/PCL microspheres was restricted or facilitated, the islands correspondingly grew inadequately or excessively (Fig. S1, ESI†). Therefore, it was concluded that the island–sea topography was the result of movement and assembly of the PLGA molecules derived from fast solidification during the emulsion process. After the oil phase was emulsified into small droplets in the aqueous phase, the solvent in a droplet would diffuse immediately from just under the surface into the aqueous phase, causing a prompt solidification of the surface prior to the internal zone. This inhibited the movement of the PLGA molecules near the surface, resulting in lateral phase separation instead of a stratified one. The PLGA molecules had to locally assemble into the islands pattern. The solvent inside the droplets diffused out gradually, providing enough time for PCL and PLGA to separate into the layered structure.

In order to figure out how the cavities inside were formed, the shrinking ratios of PCL, PLGA and PLGA/PCL microspheres were compared and the results are listed in Table 1. It is known that the oil droplets in an oil/water emulsion will shrink as the solvent evaporates. The PLGA microspheres had a larger shrinkage than the PCL microspheres during solidification. Surprisingly, the PLGA/PCL microspheres had the lowest shrinking ratio. This meant that the occurrence of phase separation might inhibit the shrinking of the oil droplets. Since the PLGA core was expected to shrink the most, this inhibiting effect induced the generation of internal cavities to compensate for the shrinking.

Table 1 The shrinking ratios of different microspheres and the contact angles of the polymer films and microsphere discs

	Shrinking ratio	Contact angle of the films	Contact angle of the discs
PLGA	2.55 ± 0.14	82.60 ± 0.36	78.92 ± 0.34
PCL	1.86 ± 0.09	108.3 ± 0.88	96.06 ± 1.95
PLGA/PCL	1.73 ± 0.04	—	89.76 ± 1.51

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Combined with the results above, it was concluded that PCL and PLGA in an oil droplet would start phase separating as the solvent evaporated. Thermodynamically, PCL and PLGA occupied the shell and the core, respectively (Fig. 5a-II). Since the separation was incomplete, some PLGA remained in the shell and some PCL remained in the core (Fig. 5a-III). The phase separation inhibited the shrinking of the PLGA core, leading to the generation of internal cavities (Fig. 5a-II and III). Since the polymer on the surface solidified faster than the internal counterpart, only lateral separation took place on the surface, forming an island–sea topography (Fig. 5b-II and III). The lateral separation started and ended earlier than the stratified separation.

Fig. 5 Schematic of the formation of the layered architecture (a) and the island–sea topography (b) in the PLGA/PCL microsphere. (a) I: Cross-section of the initial oil droplet in the aqueous phase; II: the beginning of the phase separation between PLGA and PCL and the shrinking of the oil droplet; and III: the layered architecture and the internal cavity in a solidified microsphere. (b) I: The initial oil droplet; II: the beginning of the phase separation and shrinking; and III: the island–sea topography on a solidified microsphere. Since the surface of the oil droplet solidified earlier than the internal part, the start and end of the island–sea topography was earlier than the layered architecture.

3.3 In vitro degradation of the PLGA/PCL microspheres

Fig. 6 reveals the evolution of the morphology with degradation. After one week of degradation, the morphology hardly changed (Fig. 6a). After two weeks, the PLGA islands had obviously collapsed (Fig. 6b). The PCL shell still appeared smooth. However, as the degradation proceeded to three weeks, the PLGA islands basically lost their original shapes and the entire surface became rough (Fig. 6c). After 31 days, the microsphere still remained a spherical shape. Nevertheless, the PLGA islands disappeared significantly and just left concaves (Fig. 6d).

Fig. 6 The morphologies of a microsphere after 7 (a), 14 (b), 21 (c) and 31 (d) days of in vitro degradation in PBS.

It is well accepted that PLGA generally degrades faster than PCL. The PLGA we used (50/50, 30 kDa) had a hydrophilic ratio and a low molecular weight, which contributed to the degradation of the polymer. This explained the obvious collapse of the PLGA islands after two weeks. The absence of detachment of the PLGA islands from the PCL matrix after two weeks indicated that the islands were bonded firmly to the PCL substrate. Since PCL degraded slowly, the shell remained unchanged during the whole test.

3.4 Effect of PLGA islands on the hydrophilicity of the microspheres

Although PCL is widely used as a shell in particle systems, its hydrophobicity is not desired in biomedical applications. The results of the contact angle analysis are listed in Table 1. To retain the PLGA islands, the microspheres were compressed into discs. As expected, the PCL film had an undesired high contact angle while the PLGA film was more hydrophilic. For the PLGA/PCL microspheres, the existence of the PLGA islands on the surface improved the hydrophilicity of the PCL shell (from 96.06° ± 1.95 to 89.76° ± 1.51). As the PLGA islands existed as individual zones instead of completely covering the PCL, the surface of the PLGA/PCL microsphere microscopically consisted of zones having different hydrophilicity.

3.5 Cell culture on the PLGA/PCL microspheres

Fig. 7 shows the proliferation of the mMSCs on the PLGA/PCL and PCL (as a control) microspheres. It was seen that the mMSCs grew well on both microspheres. At each time point, the PLGA/PCL and PCL microspheres basically had the same extent of proliferation. This was consistent with the results in other work.^13,22 Fig. 8 shows the morphologies of the mouse mesenchymal cells on the PCL and PLGA microspheres. On the 1^st day, some of the mMSCs had spread on both microspheres, with some still maintaining their round shape. On the PCL microsphere, the cells seemed to have more pseudopods protruding from the bodies (Fig. 8a). On the PLGA/PCL microspheres (Fig. 8b), the cells mainly maintained their spindle shape. Interestingly, it was found that the cells tended to adhere on the PLGA islands. The cells spread over one or even more islands, with a few bypassing the islands. On the 2^nd day, basically all the cells had spread well on the PCL microspheres (Fig. 8c). The mMSCs still tended to adhere on the PLGA islands (Fig. 8d). Meanwhile, the cells also tended to bridge between adjacent microspheres, which was not obviously observed in the PCL control.

Fig. 7 Proliferation of the mMSCs on the PCL and PLGA/PCL microspheres evaluated by a CCK-8 kit. The time points were 1, 3 and 5 days.

Fig. 8 SEM images of the attachment of the mMSCs on the PCL (a and c) and PLGA/PCL (b and d) microspheres after 1 (a and b) and 2 (c and d) days of culture. The top right image in (d) is the cells bridging two microspheres.

4. Discussion

So far, most of the doping efforts related to polymer composites have been conducted on planar substrates. Compared with planar substrates, microspheres are more applicable for tissue repair. However, in conventionally fabricated microspheres, even those composed of two polymers, the surfaces are composed of a single component. The doping effect could not be reflected. In this paper, we achieved doping directly on the surface of the PLGA/PCL microspheres, producing a layered architecture and an island–sea topography. As stated in detail in Section 3.2, the fast solidification rate on the surface forced the PLGA molecules to assemble locally into islands.

The existence of the islands enhanced the hydrophilicity of the PCL shell. It is generally agreed that cells prefer hydrophilic substrates.^23,24 Much effort has been taken to enhance the adhesion and osteogenic differentiation of mesenchymal cells on a PCL matrix through PLGA doping.^13,25 However, the previously prepared PLGA doped PCL substrates lacked a defined topography. Improving the macroscopic hydrophilicity was the main doping outcome. In our microspheres, the surface of the PLGA/PCL microspheres microscopically consisted of hydrophilic PLGA islands and hydrophobic PCL sea. It is reasonable to believe that when the cells attach onto a PLGA/PCL microsphere, they recognize the difference between PLGA and PCL. Since the cells prefer the hydrophilic PLGA, they migrate towards and adhere on the islands. On the other hand, because the PLGA islands are convex, the mMSCs have to climb on them, which might be unfavourable for cell adhesion. So less pseudopods protrude compared to on the plain PCL microsphere and the cells are more mobile to migrate towards and bridge between two adjacent microspheres. The two-edged role of the PLGA islands explains why the PLGA/PCL with better hydrophilicity did not show a higher cell proliferation compared to the PCL control. Further work is required to regulate the island–sea topography and explore the cellular response.

5. Conclusion

This work presents a PLGA/PCL composite microsphere with both a layered architecture and an island–sea topography. The novel structure is the outcome of externally lateral and internally stratified phase separation between PLGA and PCL. The mMSCs grew well on the island–sea surface. Moreover, the existence of the more hydrophilic PLGA islands made the mMSCs exhibit a special adhesion behaviour. This study may be meaningful in designing microspheres for cell-contacting tissue repair.

Acknowledgements

This work was financially supported by the National Basic Research Program of China (2012CB619100, 2011CB606204), the National Natural Science Foundation of China (51372085), the Science Foundation of Guangdong Province (S2012010009188), the 111 project (B13039) and the Fundamental Research Funds for the Central Universities (2013ZZ0005).

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Footnote

† Electronic supplementary information (ESI) available: SEM images of the PLGA/PCL microspheres with an increased total concentration of PLGA and PCL and a slowed rate of solvent evaporation are shown in Fig. S1. The GPC analysis of the degraded microspheres is shown in Table S1. See DOI: 10.1039/c3ra45274c

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