Fabrication of Poly Caprolactone (PCL) Based Microspheres for Drug Delivery and Tissue Engineering Application

NYOKE Goon Chiaa, NAZNIN Sultanab,*

Department of Clinical Sciences, FBME Universiti Teknologi Malaysia 81310 Skudai, Johor

achianyokegoon@gmail.com , bnaznin@biomedical.utm.my

Keywords: Microspheres; Polyvinyl alcohol (PVA); Poly (Caprolactone) (PCL); Tissue Engineering, Scanning Electron Microscopy (SEM) Abstract. This paper reports the fabrication of poly (caprolactone) (PCL) based biodegradable

microspheres using freeze-drying technique for drug delivery and tissue engineering application.

Microspheres play an important role in tissue engineering and drug delivery applications. The

properties can determine the success or failure of the development of the materials. Biodegradable

and biocompatible synthetic polymer, PCL was used to fabricate biodegradable microspheres in this

study. Through a double emulsion and freeze-drying technique, the microspheres were produced.

Different concentrations of polymers were used to fabricate the microspheres. The microspheres

were characterized using different techniques.

Introduction

A variety of biodegradable microspheres have been studied for regeneration of tissue as an

important factor of tissue engineering. The ideal biomaterial for the drug delivery and tissue

engineering is expected to support cell attachment, have very good mechanical properties, be

fabricated easily into a desired shape, allows controlled release of biodegrade materials to patient

[1]. Biodegradable microspheres have attracted more interest due to its various applications in the

controlled release of growth factor [2] and drug delivery [3] in tissue engineering. However, they

cannot give good support for cell and tissue as a delivery system with good biocompatibility over

long time ago.

Poly (Caprolactone) (PCL) is a biodegradable and biocompatible polymer that had been used in

various biomedical applications such as bone grafting, tissue engineering and drug delivery system

[4-6]. It has limited usage in certain application and it is hydrophobic [7-9] .This scenario can be

change by mixing soluable polymer (PVA) by giving hydrophilicity to support its property. By

hydrolysis, PCL degrades by decreasing its ester bonds in physiological conditions in the human

body. It has slower degradation rate compared to polylactide and faster than polyhydroxy butyrate

(PHB) polymers. It has been studied for as a scaffold for tissue engineering. PVA is an atactic

resource but displays crystalline as the hydroxyl groups are tiny to fit into the lattice by not

disrupting it. The property of this material includes emulsifying, adhesive properties, low cost,

nontoxic and biocompatibility [8-11].

Freeze-drying technique is known as lyophilization [12, 13]. It is one of the techniques to get the

microspheres with suitable properties to be used in tissue engineering application. The purpose of

this study was to fabricate PCL based biodegradable microspheres for drug delivery and tissue

engineering application.

Materials and methods

Material of Microspheres. PCL (molecular weight 70000-90000) and Poly (venyl alcohol (PVA)

(molecular weight 85000-129000, 88% hydrolyzed) were purchased from Sigma. All the chemicals

were purchased from Sigma also.

Preparation of Microspheres. The Poly Caprolactone (PCL) based microspheres were prepared by

freeze-drying technique in laboratory. In brief, PVA aqueous solution was poured into a PCL

solution in dichloromethane. Then, the mixture was stirred under a magnetic stirrer into beaker for

24 hours (800rpm) at room temperature. After stirring, the mixture were washed and filtered in a

clear beaker using filtering paper. The powder in the filter paper were washed again with 100ml

distil water for filtering purpose. The powder was placed in plastic container for 24 hours into a

freezer. After being frozen for one day, it was placed into the freezer-drying vessel (Labconcco-

Freeze- dry system) for 72 hours for lyophilization. The final products were stored in a dissiccator.

In order to determine the size of microspheres, all of the samples were characterized by Scanning

Electron Microscope (SEM).

Characterization of Biodegradable Microspheres. Analysis had been carried out to characterize

biodegradable microspheres. Scanning electron microscopy (SEM) was used to examine the

morphology and characteristic of the samples. The freeze-dried samples were put on aluminum

sample mount without coating and viewed under SEM. An accelerating voltage of 15 KV was used

for examining surface morphology identification. The sizes and average pore of microspheres were

also determined by frequency distribution on the size of the spheres using SEM.

In Vitro Degradation Study. Sample A and sample C were taken for in vitro degradation study.

First of all, each of the samples was weighted 0.1g respectively. Each sample was placed in a

polyethylene tube containing 8mL of phosphate buffered saline (PBS, pH 7.4). The tubes were

placed in a shaking water bath at 37 ◦C for one month. At predetermined time points, the release

medium was completely withdrawn and replaced with 8 mL of fresh PBS. Both of the samples were

characterized by Scanning Electron Microscope (SEM) in order to exanimate the changes size of

pore microspheres.

Results and Discussions

PCL Microspheres.

Fig. 1: SEM micrographs and size distribution of all samples

In this study, PCL microspheres were fabricated. Sample A was

PCL/PVA meanwhile sample B was prepared using 3.3%/0.8% of PCL/PVA. Sample C was

prepared using 10%/0.20% of PCL/PVA and sample D was prepared using 20%/0.5% of

PCL/PVA. Figure 1 presents the SEM of all of the samples and the siz

the microspheres had a smooth surface and the average sizes of all samples were calculated. The

histograms show the frequency distribution of the siz

sample based on the result of the frequency distribution on the sizes of the spheres for all samples.

Sample A provided the largest average size of microspheres which was 33.55

microspheres were not uniform in sizes. On the other hand, sample B had the second largest average

size of microspheres which was 23.41

µm. Sample C provided the smallest diameter which was 7.62

relatively homogeneous in sizes.

In Vitro Degradation Study.

(a)

(c)

Figure 2: Sample A before (a) and after (b) in vitro degradation

After in vitro degradation of 2 months in

smaller compare to the initial microspheres (Figure 2). Some broken spheres were also observed in

Figure 2b. PCL microspheres exhibited slow degradation after in vitro biodegradation.

sample C also had some smaller diameter microspheres after in vitro degradation for 2 months in

PBS. Some micropores were also observed after in vitro degradation.

showed the similar changes on the size distribution and some broken spheres

SEM.

Conclusions

Poly Caprolactone (PCL) based microspheres were prepared using double emulsion and freeze

drying technique. The biodegradable microspheres were prepared at different PCL concentration

ratio. Sample C provided the best result among the samples in this study as the spheres w

distributed uniformly with much smallest diameter of 7.62

the microspheres became smaller in sizes. Some broken spheres and micropores in the spheres were

In this study, PCL microspheres were fabricated. Sample A was prepared using 6%/0.12% of

PCL/PVA meanwhile sample B was prepared using 3.3%/0.8% of PCL/PVA. Sample C was

prepared using 10%/0.20% of PCL/PVA and sample D was prepared using 20%/0.5% of

Figure 1 presents the SEM of all of the samples and the sizes of the microspheres. All

the microspheres had a smooth surface and the average sizes of all samples were calculated. The

histograms show the frequency distribution of the size of the sphere. The average pore size of each

e frequency distribution on the sizes of the spheres for all samples.

Sample A provided the largest average size of microspheres which was 33.55

microspheres were not uniform in sizes. On the other hand, sample B had the second largest average

of microspheres which was 23.41 µm. Sample D had the average size of microsphere of

Sample C provided the smallest diameter which was 7.62 µm. The microspheres were

(b)

) (d)

Sample A before (a) and after (b) in vitro degradation; Sample C before (c) and after (d

in vitro degradation

After in vitro degradation of 2 months in PBS, the sizes of microspheres were observed to be

smaller compare to the initial microspheres (Figure 2). Some broken spheres were also observed in

Figure 2b. PCL microspheres exhibited slow degradation after in vitro biodegradation.

lso had some smaller diameter microspheres after in vitro degradation for 2 months in

PBS. Some micropores were also observed after in vitro degradation. In brief, both the samples

showed the similar changes on the size distribution and some broken spheres which was revealed by

(PCL) based microspheres were prepared using double emulsion and freeze

drying technique. The biodegradable microspheres were prepared at different PCL concentration

ratio. Sample C provided the best result among the samples in this study as the spheres w

distributed uniformly with much smallest diameter of 7.62 µm. After the in vitro degradation study,

the microspheres became smaller in sizes. Some broken spheres and micropores in the spheres were

prepared using 6%/0.12% of

PCL/PVA meanwhile sample B was prepared using 3.3%/0.8% of PCL/PVA. Sample C was

prepared using 10%/0.20% of PCL/PVA and sample D was prepared using 20%/0.5% of

es of the microspheres. All

the microspheres had a smooth surface and the average sizes of all samples were calculated. The

he average pore size of each

e frequency distribution on the sizes of the spheres for all samples.

Sample A provided the largest average size of microspheres which was 33.55 µm. The

microspheres were not uniform in sizes. On the other hand, sample B had the second largest average

average size of microsphere of 17.63

µm. The microspheres were

Sample C before (c) and after (d)

PBS, the sizes of microspheres were observed to be

smaller compare to the initial microspheres (Figure 2). Some broken spheres were also observed in

Figure 2b. PCL microspheres exhibited slow degradation after in vitro biodegradation. Similarly,

lso had some smaller diameter microspheres after in vitro degradation for 2 months in

In brief, both the samples

which was revealed by

(PCL) based microspheres were prepared using double emulsion and freeze-

drying technique. The biodegradable microspheres were prepared at different PCL concentration

ratio. Sample C provided the best result among the samples in this study as the spheres were

the in vitro degradation study,

the microspheres became smaller in sizes. Some broken spheres and micropores in the spheres were

also observed which can indicate the degradation of microspheres.

Acknowledgment

The author would like to thank Faculty of Bioscience and Medical Engineering, Univesiti

Teknologi Malaysia (UTM) for the facilities and support. N. Sultana acknowledges the financial

support from Ministry of Higher education and RMC for research grants GUP (vot: 05H07).

References

[1] D.H. Kempen, L. Lu, X Zu, W.J. Dhert, B.L. Currier and M.J. Yaszemski: Controlled drug

release from a novel injectable biodegradable microsphere/scaffold composite based on

poly(propylene fumarate. J. Biomed. Mater. Res. A Vol. 7 (2006), p. 103-111

[2] A.J. DeFail, C.R Chu, N. Izzo and K.G. Marra: Controlled release of bioactive TGF-beta 1 from

microspheres embedded within biodegradable hydrogels. Biomat. Vol. 27 (2006), p. 1579-1585

[3] B.S. Zolnik, P.E. Leary and D.J. Burgess: Elevated temperature accelerated release testing of

PLGA microspheres. J. Cont. Release Vol. 112 (2006), p. 293-300

[4] W.L. Suchane, P. Shuk, K. Byrappa, R.E. Riman, K.S. TenHuisen and V.F. Janas:

Mechanochemical-hydrothermal synthesis of carbonated apatite powders at room temperature.

Biomat. Vol. 23 (2002) p. 699-710

[5] M. Labet and W. Thielemans: Synthesis of polycaprolactone: a review. Chem. Soc. Rev. Vol. 38

(2009), p. 3484-3504

[6] T. Kean, S. Roth and M. Thanou: Trimethylated chitosans as non-viral gene delivery vectors:

cytotoxiticy and transfection efficiency. J. Cont. Release Vol. 103 (2005), p. 643-653

[7] N. Bhattarai, Z. Li, J. Gunn, M. Leung, A. Cooper, D. Edmondson, O. Veiseh, M.-H. Chen, Y.

Zhang, R.G. Ellenbogen and M. Zhang: Natural-synthetic polyblend nanofibers for biomedical

applications. J. Adv. Mat. Vol. 21 (2009), p. 2792-2797

[8] X. Yang, X. Chen and H. Wang: Acceleration of osteogenic differentiation of preosteoblastic

cells by chitosan containing nanofibrous scaffolds. J. Biomacromolecules Vol. 8 (2007), p.

807-816

[9] F. Roozbahani, N. Sultana, A. F. Ismail, and Hamed Nouparvar, Effects of Chitosan Alkali

Pretreatment on the Preparation of Electrospun PCL/Chitosan Blend Nanofibrous Scaffolds for

Tissue Engineering Application, Journal of Nanomaterials. 2013(2013), Article ID 641502, 1-6

doi:10.1155/2013/641502

[10] A.K. Moghe, R. Hufenus, S.M. Hudson and B.S. Gupta: Effect of the addition of a fugitive salt

on of polymer. Polymer Vol. 50 (2009), p. 3311-3318

[11]N. Sultana and T. H. Khan: Water Absorption and Diffusion Characteristics of

Nanohydroxyapatite (nHA) and Poly(hydroxybutyrate-co-hydroxyvalerate-) Based Composite

Tissue Engineering Scaffolds and Nonporous Thin Films. J Nanomater, vol. 2013, Article ID

479109, p.1-8 (2013). doi:10.1155/2013/479109

[12] N. Sultana and M. Wang: PHBV/PLLA-based composite scaffolds fabricated using an

emulsion freezing/freeze-drying technique for bone tissue engineering:surface modication and

in vitro biological evaluation. Biofabrication Vol. 4 (2012)

[13] N. Sultana and T.H. Khan: In Vitro degradation of PHBV scaffold and nHA/PHBV composite

scaffolds containing hydroxyapatite nanoparticle for bone tissue engineering. J. Nanomater.

Vol. 2012 (2012)