Optimization of ultraviolet ozone treatment process for improvement of polycaprolactone (PCL) microcarrier performance

Abstract

Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the surface functionality of certain polymeric microcarriers for effective cell attachment and growth remains a challenge. Polycaprolactone (PCL), a biodegradable polymer has received considerable attention due to its good mechanical properties and degradation rate. The drawback is the non-polar hydrocarbon moiety which makes it not readily suitable for cell attachment. This report concerns the modification of PCL microcarrier surface (introduction of functional oxygen groups) using ultraviolet irradiation and ozone (UV/O₃) system and investigation of the effects of ozone concentration, the amount of PCL and exposure time; where the optimum conditions were found to be at 60,110.52 ppm, 5.5 g PCL and 60 min, respectively. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.92 nmol/g and the amount of gelatin immobilized was 320 ± 0.9 µg/g on UV/O₃ treated microcarriers as compared to the untreated (26.83 ± 3 µg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with the attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93°-49.34°) after UV/O₃ treatment and subsequently after immobilization of gelatin. The attachment and growth kinetics for HaCaT skin keratinocyte cells showed that adhesion occurred much more rapidly for oxidized surfaces and gelatin immobilized surface as compared to untreated PCL.

Keywords: Gelatin immobilization; Microcarrier; Polycaprolactone (PCL); Surface modification; Ultra violet ozone (UV/O3).

Fig. 1

Amount of carboxylic acid (COOH) functional group introduced on the surface of untreated microcarrier and microcarrier treated with either UV/O₃treatment, ozone aeration (only) treatment, UV irradiation (only) treatment, respectively. Oxygen flow rate (2 lpm), sample amount (1 g), exposure time (10 min) and UV intensity (22 mW/cm²) were held constant for the respective treatment

Fig. 2

Interaction between carboxyl (COOH) concentration on microcarrier surface and amount of gelatin immobilized. Kim et al. (2009) found that carboxyl functional group increased proportionally with the exposure time of oxygen plasma treatment. Subsequently, the efficiency of the protein immobilization on the treated surface was increased thus generating functional amine (NH₂) group for cell adhesion

Fig. 3

3-D response surface and 2-D contour plot of interaction between a ozone concentrations and amount of samples; b exposure time and ozone concentration; c exposure time and amount of samples

Fig. 4

ATR-FTIR spectra of untreated PCL, UV/O₃ treated PCL (UV/O₃ PCL) and gelatin coated PCL (GEL PCL)

Fig. 5

SEM images show the untreated PCL microcarrier (a), UV/O₃ treated PCL microcarrier (b), and gelatin coated PCL microcarrier (c). d, e and f are higher magnification of (a–c), respectively

Fig. 6

Growth kinetics of human keratinocytes cells (HaCaT) on different microcarriers cultured in stirred spinner vessels (x) UV/O₃ PCL, (filled circle) gelatin immobilized, (filled diamond) untreated PCL. Result were based on three independent experiment (n = 3, mean ± SD)

Fig. 7

Micrograph of HaCaT at 96 h on a untreated PCL, b UV/O₃ PCL, c Gelatin coated PCL visualized using an inverted phase contrast microscope. SEM image of d HaCaT cells on UV/O₃ PCL and e HaCaT cells on gelatin-coated PCL