ARTICLE pubs.acs.org/bc
PCL Film Surfaces Conjugated with P(DMAEMA)/Gelatin Complexes for Improving Cell Immobilization and Gene Transfection C. Y. Li,‡,† W. Yuan,§,† H. Jiang,‡ J. S. Li,|| F. J. Xu,*,‡ W. T. Yang,‡ and J. Ma*,§ ‡
)
State Key Laboratory of Chemical Resource Engineering, Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing 100029 China § State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing 100021 China College of Polymer Science and Engineering, Sichuan University, Chengdu 610065 China ABSTRACT: Successful gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration by enabling the localized production of therapeutic drugs. Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but its unfavorable cell-adhesion property needs to be improved. In this work, the PCL film surface was conjugated with poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA))/gelatin complexes via surface-initiated atom transfer radical polymerization (ATRP) for improving cell immobilization and subsequent gene transfection. A simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL film. Well-defined P(DMAEMA) brushes were subsequently prepared via surfaceinitiated ATRP from the initiator-functionalized PCL surfaces. The P(DMAEMA) chains with a pKa of 7.07.3 were used for conjugating gelatin with a pI of 4.7 via electrostatic interaction. The amount of complexed gelatin increased as that of the grafted P(DMAEMA) layer. The cell-adhesion property on the functionalized PCL surface could be controlled by adjusting the ratio of P(DMAEMA)/gelatin. It was found that the gene transfection property on the immobilized cells was dependent on the density of the immobilized cells on the functionalized PCL film. With the good cell-adhesive nature of gelatin and the efficient gene transfection on the dense immobilized cells, the incorporating the suitable of P(DMAEMA)/gelatin complexes onto PCL surfaces could endow the PCL substrates new and interesting properties for potential tissue engineering applications.
’ INTRODUCTION Polycaprolactone (PCL) has been widely adopted for biomaterials and biomedical applications because of its slow degradability and good biocompatibility, as well as its good mechanical and thermoplastic characteristics.1,2 The application of synthetic biomaterials depends upon their interfacial properties and resultant interactions with cells and biological fluids in vivo. The ability to manipulate and control the surface properties of a biomaterial without altering its bulk properties is of crucial importance in the designing of biomedical materials.3,4 Covalent tethering of well-defined polymer brushes on a solid substrate is an effective method for modifying the surface properties.57 Surface-initiated atom transfer radical polymerization (ATRP) allows the preparation of well-defined dense polymer brushes and hence provides the high capacity for conjugating functional molecules.79 Recently, we explored surface-initiated ATRP of glycidyl methacrylate (GMA) to tailor the functionality of polycaprolactone (PCL) film surfaces.10 The well-defined PGMA brushes were used for conjugating cell-adhesive collagen and RGDS to improve the cell-adhesion properties of the PCL film surface. r 2011 American Chemical Society
The immobilization of ATRP initiators on PCL surfaces was based on the hydroxyl groups from the surface hydrolysis of PCL.10 However, such hydrolysis process of polyester also produced the corresponding COOH groups, which could hamper the immobilization efficiency of the ATRP initiators. Further improvement in cell adhesion properties of the PCL film surface will facilitate its biomedical applications. In addition, the expanded research about the possibility to combine the immobilized cells with other functionalities such as local gene delivery is also very important to the development of multifunctional PCL-based tissue scaffolds. It was reported that successful gene transfection on a biomaterial could enable the localized production of therapeutic drugs to facilitate tissue repair and regeneration.11,12 In this work, a simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL surfaces (Figure 1). It was well-known that the aminolysis process of polyester film surfaces with 1,6-hexanediamine can Received: May 10, 2011 Revised: August 10, 2011 Published: August 18, 2011 1842
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Figure 1. Schematic diagram illustrating the reaction of free amine and hydroxyl groups on the aminolyzed PCL film surface with 2-bromoisobutyrate bromide (BIBB) to produce the PCL-Br surface, surface-initiated ATRP of DMAEMA from the PCL-Br surface to produce the PCL-g-P(DMAEMA) surface, and physical immobilization of gelatin to produce the PCL-g-P(DMAEMA)/gelatin surface for biomedical applications.
introduce the free amino and hydroxyl groups.1315 Both reactive groups are ready to react with 2-bromoisobutyryl bromide to produce the ATRP initiator-coupled surfaces (the PCL-Br surfaces). Instead of P(GMA) brushes,10 well-defined poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA)) brushes (the PCL-g-P(DMAEMA) surfaces) were subsequently prepared via surface-initiated ATRP of DMAEMA from the PCL-Br surfaces. The P(DMAEMA) chains with a pKa of 7.07.316,17 were used for conjugation of gelatin with a pI of 4.718 (derived from naturally extracellular matrix collagen) via electrostatic interaction for improving the cell-adhesion properties of the PCL surfaces. The subsequent gene transfection in the immobilized cells was further investigated to develop multifunctional PCL-based scaffolds. The chemical compositions, topography, cell adhesion, and gene delivery characteristics of the modified PCL film surfaces were characterized by X-ray photoelectron spectroscopy (XPS), atomic force spectroscopy (AFM), fluorescent microscopy, and fluorescence-activated cell sorting analysis (FACS), respectively.
’ EXPERIMENTAL PROCEDURES Materials. Polycaprolactone pellets (PCL, Mn = 42 500), 1,6hexanediamine, branched polyethylenimine (PEI, Mw ∼25 000 Da), gelatin powder from porcine skin, 2-bromoisobutyrl bromide (98%), (2-dimethylamino)ethyl methacrylate (DMAEMA, >98%), copper(I) bromide (CuBr, 99%), copper(II) bromide (CuBr2, >98%), and 2,20 -byridine (Bpy, 98%), were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. DMAEMA was used after removal of the inhibitors in a ready-to-use disposable inhibitor-removal column (Sigma-Aldrich). HEK293 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Aminolysis of PCL Films. The polycaprolactone (PCL) film was prepared by dissolving 4 g of PCL pellets in 40 mL of methylene chloride. The polymer solution was then cast onto a glass substrate and the solvent was removed by controlled evaporation at room temperature over a period of 24 h. The so-obtained pristine PCL film with a thickness of 40100 μm was washed with copious amounts of an alcohol/water (1/1, v/v) mixture.10 The dried PCL films were cut into specimen size of
2 cm 2 cm for the subsequent surface reactions. The cleaned PCL films were subsequently immersed in 1,6-hexanediamine/ 2-propanol solution of 60 mg/mL for 2 h at room temperature. The aminolysis process produced the PCL-NH2 films. Surface-Initiated ATRP of DMAEMA and Gelatin Immobilization. As shown schematically in Figure 1, the immobilization of ATRP initiator on the PCL-NH2 film surface was accomplished via the reaction of the amino and hydroxyl groups with 2-bromoisobutyrate bromide (BIBB). The PCL-NH2 films were introduced into 10 mL of dried hexane, followed by addition of 0.5 mL of 2-bromoisobutyryl bromide and 0.2 mL of dry pyridine. The reaction mixture was kept for 2 h at 0 °C, and then at room temperature for 22 h to produce the 2-bromoisobutyrylimmobilized PCL surface (the PCL-Br surface). The PCL-Br substrate was washed repeatedly with a methanol/water (1/2, v/v) mixture and dried by pumping under reduced pressure. In order to estimate the ATRP initiator (alkyl bromide) density of the PCL-Br surface, the initiator density was first set at an unknown value, F* (Brs/nm2). On the basis of the initiator density (F*), mass density (F1) of the 2-bromoisobutyrate (BIB) layer (1.0 g/cm3), and molecular weight (M1) of BIB (165.5 g/mol), the thickness (h) of the initiator layer was estimated to be about 0.27F* (h = F* 3 M1/F1). From the mass density (F2) of the PCL film (1.0 g/cm3), the molecular weight (M2) of the caprolactone (CL) repeat unit (114 g/mol), the stoichiometry (C6H10O2) of the CL repeat unit, and the sampling depth (about 7.5 nm in an organic matrix) of the XPS technique, the number (n) of the total C atoms per unit volume of the XPS probing depth (V = 7.5 nm3) is about (237.8 4.6F*) (n = 4F* + 6[(7.5 h)F2/M2]).10 On the basis of the [Br]/[C] ratio (r) (determined from the sensitivity factor-corrected Br 3d and C 1s core-level spectral area ratio of the PCL-Br surface), the initiator density (F*) could be calculated by r = F*/n. For the preparation of P(DMAEMA) brushes on the PCL-Br surface, the reaction was carried out using a [DMAEMA (5 mL)]/[CuBr]/[CuBr2]/[2,20 -byridine, Bpy] molar feed ratio of 100:1:0.2:2 in 10 mL of methanol/water mixed solvent (1/4, v/v) at room temperature in a Pyrex tube. The mixture was stirred and degassed with argon for 20 min. The PCLBr substrate was then introduced into the reaction mixture. The reaction was allowed to proceed for 5 to 120 min to produce the 1843
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Table 1. Reaction Time, Grafting Yield (GY), Chemical Composition, and Static Water Contact Angle of the Functionalized PCL Surfaces water contact sample Pristine PCL PCL-NH2a PCL-Br PCL-g-P(DMAEMA)1 b
reaction
GYd
angle
time
(μg/cm2)
((3°)
-- -24 h 24 h -5 min 0.66 ((0.2)
70 65 82 58
PCL-g-P(DMAEMA)2 b
30 min 3.7 ((0.6)
49
PCL-g-P(DMAEMA)3 b
60 min 7.7 ((0.5)
43
PCL-g-P(DMAEMA)1/Gelatin c
48 h 0.7 ((0.1)
49
PCL-g-P(DMAEMA)2/Gelatin c
48 h 3.2 ((0.3)
46
PCL-g-P(DMAEMA)3/Gelatin c
48 h 6.1 ((0.7)
42
Obtained by immersing the pristine PCL film in 1,6-hexanediamine/2propanol solution of 60 mg/mL for 24 h at room temperature. b Reaction conditions: [DMAEMA]/[CuBr]/[CuBr2]/[Bpy] = 100:1:0.2:1.2 in methanol/water mixture (1/4, v/v) at room temperature. c Obtained by immersing the corresponding PCL-g-P(DMAEMA) surfaces in PBS solution containing the gelatin at a concentration of 10 mg/mL at 37 °C. d Grafting yield (GY) is defined as GY = (Wa Wb)/A, where Wa and Wb represent the weight of the dry films after and before grafting, respectively, and A is the film area (about 28 cm2). Data are presented as means ( standard deviation (n = 3). a
PCL-g-P(DMAEMA) surface. After the reaction, the PCL-gP(DMAEMA) surface was washed thoroughly with methanol and water to ensure the complete removal of the physically adsorbed reactants, prior to being dried under reduced pressure. Details on the surface-initiated ATRP had been described earlier.19 In this work, gelatin was immobilized onto the PCL-g-P(DMAEMA) surface via electrostatic interaction to improve the cell-adhesion properties of the PCL film surface. The PCLg-P(DMAEMA) surfaces were immersed into PBS solution (pH 7.4) containing the gelatin at a concentration of 10 mg/mL at 37 °C. The electrostatical interaction was allowed to proceed for 48 h to produce the PCL-g-P(DMAEMA)/Gelatin surface. After the immobilization reaction, the films were washed sequentially with doubly distilled water at 40 °C to desorb the reversibly bound gelatin. These functionalized PCL surfaces were finally washed with sterilized PBS for further biological experiments. It is very difficult to estimate the fractional surface coverage with gelatin. In this work, the surface concentration of immobilized gelatin was instead expressed as the [N]/[C] ratio (determined from the sensitivity factors-corrected N 1s and C 1s core-level spectral area ratio of the PCL-g-P(DMAEMA)/Gelatin surfaces. Surface Characterization. The chemical compositions of the modified PCL film surfaces were characterized by X-ray photoelectron spectroscopy (XPS). The XPS measurements were performed on a Kratos AXIS HSi spectrometer using a monochromatized Al Kα X-ray source (1486.6 eV photons) and procedures similar to those described earlier.19 The static water contact angles of the pristine and functionalized PCL film surfaces were measured at 25 °C and 60% relative humidity, using the sessile drop method with 3 μL water droplets, in a telescopic goniometer (Rame-Hart model 100-00-(230), manufactured by Rame-Hart, Inc., Mountain Lakes, NJ). The topography of the modified PCL surfaces was studied by atomic force
microscopy (AFM), using a Dimension 3100 AFM from the Veeco-Digital Instruments (Santa Barbara, CA, USA). In each case, an area of 40 μm 40 μm square was scanned using the tapping mode. The drive frequency of the equipment, with the voltage between 3.0 and 4.0 V, was 330 ( 50 kHz. The drive amplitude was about 300 mV and the scan rate was 0.51.0 Hz. The arithmetic mean of the surface roughness (Ra) reported was calculated from the roughness profile determined by AFM. Cell Culture. The cell-adhesion and gene transfection characteristics of the functionalized PCL surfaces were assessed by HEK293 cell lines. Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. All media contained 100 U/mL of penicillin and 100 μg/mL of streptomycin. Cells were incubated at 37 °C and supplemented with 5% CO2 in the humidified chamber. For assaying the cell-adhesion characteristics of the functionalized PCL films, the surface-modified PCL films were washed twice with PBS, prior to being placed into the wells of a 24-well culture plate. HEK293 cells at a density of 8 104 cells/well in culture media were seeded and cultured on the functionalized PCL films for 24 h. The surfaces after incubation were washed twice with PBS solution to remove the dead and loosely attached cells. The cultured cells on the film surfaces were fixed with cold 70% ethanol at 4 °C for 2 h and then stained for 15 min with 50 μg/mL propidium iodide (PI) for fluorescent imaging (BX51, Olympus; Japan). For cell number on each type of surface, the surfaces prewashed with PBS solution were incubated with trypsin EDTA solution (0.25% trypsin, 1 mM EDTA) for 10 min at 37 °C to detach the cells. The detached cells were collected and counted using a hemocytometer. For assaying the gene transfection in the immobilized HEK293 cell lines, the plasmid (encoding enhanced green fluorescent protein (EGFP)) used in this work was pEGFPC1. HEK293 cells at a density of 8 104 cells/well were seeded and cultured on the functionalized PCL films for 24 h. The PEI/ pEGFP-C1 complexes (20 μL/well containing 0.8 μg of pDNA) at the optimal N/P ratio of 10 were prepared by mixing the equal volumes of PEI and pEGFP solutions, followed by vortexing and incubation for 30 min at room temperature. At the time of transfection, the medium in each well was replaced with 300 μL of fresh normal medium (supplemented with 10% FBS). The complexes were added into the transfection medium and incubated with the cells for 6 h under standard incubator conditions. Then, the medium was replaced with 800 μL of the fresh normal medium. The cells were further incubated until a total transfection time of 48 h. The transfected cells were imaged by using an Olympus fluorescence microscope (BX51, Olympus; Japan). EGFP was excited at 488 nm and measured at 525 nm. The percentage of the EGFP positive cells was determined using fluorescence-activated cell sorting analysis (FACS). Briefly, transfected cells were washed twice with PBS and incubated with trypsinEDTA solution (0.25% trypsin, 1 mM EDTA) until cells detached from the surface. Then, 1 mL of complete medium was added to inhibit trypsin, followed by centrifugation at 1000 rpm for 3 min. The supernatant was removed and the cells were washed with PBS and resuspended in 0.8 mL PBS solution. Flow cytometry (FCM) analysis was conducted using Epics Elite ESP with excitation wavelength of 488 nm (Beckman Coulter, USA). The percentage of transfected cells was obtained by determining the statistics of cells fluorescing above the control level, where nontransfected cells were used as the control. For each sample, 10 000 cells were counted. 1844
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Figure 3. Dependence of the graft yield (GY) of the P(DMAEMA) chains of the PCL-g-P(DMAEMA) surfaces from the PCL-Br surfaces on the surface-initiated ATRP time.
Figure 2. C 1s core-level spectra of the (a) pristine PCL, (b) PCL-NH2, and (c) PCL-Br, surfaces, (b0 ) N 1s core-level spectrum of the PCL-NH2 surface, and (c0 ) Br 3d core-level spectrum of the PCL-Br surface.
’ RESULTS AND DISCUSSION Immobilization of the ATRP Initiators. For the preparation of polymer brushes, the initiator immobilization on the PCL film surface is indispensable. It was reported that the immobilization of ATRP initiators on PCL surfaces could be based on the hydroxyl groups from the surface hydrolysis of PCL.10 However, the accompanying COOH groups from the hydrolysis process could hamper the immobilization efficiency of the ATRP initiators. In this work, an alternative aminolysis-based method was developed for the covalent immobilization of ATRP initiators on the PCL surfaces. It is possible to introduce free amino and hydroxyl groups on PCL film surfaces through the aminolysis reaction with 1,6-hexanediamine.13,14 One amine group can react the with COO species to produce the covalent bond, CONH, and the other NH2 group remains free. In addition, this aminolysis process also produces the hydroxyl-terminated chains. Both the resultant NH2 and OH groups are ready to react with 2-bromoisobutyryl bromide (BIBB) to produce the ATRP initiator species (Figure 1). The pristine PCL film was immersed in 1,6-hexanediamine/2-propanol solution to induce the surface aminolysis processes for 24 h, producing the aminolyzed PCL-NH2 film (Table 1). The resultant amino and hydroxyl groups on the PCL-NH2 film surfaces were used to react with BIBB to produce the corresponding ATRP initiatorcoupled surfaces (the PCL-Br surfaces). The chemical compositions of the PCL film surfaces at various stages of surface modification were determined by X-ray photoelectron spectroscopy (XPS). Figure 2 shows the C 1s core-level spectra of the (a) pristine PCL, (b) PCL-NH2, and (c) PCL-Br surfaces. The C 1s core-level spectrum of the PCL surface can be
curve-fitted into three peak components with binding energies (BEs) at about 284.6, 286.4, and 288.7 eV, attributable to the CH, CO, and OdCO species, respectively.21 After aminolysis, the C 1s core-level spectrum of the PCL-NH2 surface possesses an additional peak at about 285.7 eV, attributable to the CN species,21 which is associated with the aminolysis process. The corresponding N 1s core-level spectrum of the PCL-NH2 surface was shown in Figure 2b0 . In this work, further prolonging the aminolysis time after 24 h at the present conditions did not lead enhancement in the intensities of the resultant N 1s signals. After the immobilization of alkyl bromide, the weak OdCN peak component with BE at about 287.5 eV, associated with the linkages between the PCL film surface and ATRP initiators, can be observed in the C 1s core-level spectrum of the PCL-Br surface. The corresponding Br 3d signal at BE of about 69 eV, characteristic of covalently bonded bromine21 was shown in Figure 2c0 . The above results confirmed that the ATRP initiators had been successfully introduced onto the PCL-Br film surface. On the basis of the [Br]/[C] ratio (r) of 1.3 102 (Figure 2c0 ), the initiator density (F*) was finally estimated to be about 2.9 initiators/nm2, higher than that (1.9 initiators/nm2) from the surface hydrolysis method.10 In addition, the static water contact angle has increased from about 70° for the pristine surface to about 82° for the PCL-Br surface (Table 1). The variation in static water contact angles for the PCL-Br surface also suggests that the alkyl halide initiators had been successfully introduced onto the PCL film surface. Surface-Initiated ATRP of DMAEMA. The physicochemical properties of PCL surfaces can be tuned by the choice of functional vinyl monomers. Instead of P(GMA) brushes,10 well-defined poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA)) brushes (the PCL-g-P(DMAEMA) surfaces) were subsequently prepared in this work via surface-initiated ATRP of DMAEMA from the PCL-Br surfaces for immobilizing biomolecules. For surface-initiated ATRP, an excess amount of deactivating Cu(II) complex (CuBr2) was added.10 The reaction conditions and the definition of grafting yield (GY) were described in Table 1. The kinetics of P(DMAEMA) growth from the PCL-Br surfaces via surface-initiated ATRP was investigated in Figure 3. An approximately linear increase in GY of the grafted P(DMAEMA) chains with polymerization time was observed, indicating that the chain growth from the PCL-Br surface was consistent with a “controlled” and well-defined process. Figure 4 shows the C 1s core-level spectra of the (a) PCL-gP(DMAEMA)1 (from 5 min of ATRP), (b) PCL-g-P(DMAEMA)2 (from 30 min of ATRP), and (c) PCL-g-P(DMAEMA)3 (from 1845
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Figure 4. C 1s core-level spectra of the (a) PCL-g-P(DMAEMA)1, (b) PCL-g-P(DMAEMA)2, (c) PCL-g-P(DMAEMA)3, (a0 ) PCL-g-P(DMAEMA)1/Gelatin, (b0 ) PCL-g-P(DMAEMA)2/Gelatin, and (c0 ) PCL-g-P(DMAEMA)3/Gelatin surfaces.
60 min of ATRP) (Table 1). The C 1s core-level spectra were composed of four peak components with BEs at about 284.6, 285.5, 286.2, and 288.4 eV, attributable to the CH, CN, CO, and OdCO species, respectively.21 In comparison with the C 1s core-level spectrum (Figure 2c) of PCL-Br, the significant increase in intensities of the CN species is consistent with the presence of P(DMAEMA) brushes on the PCL-gP(DMAEMA) surfaces. With the increase in ATRP time, the surface coverage of P(DMAEMA) brushes is expected to increase. For the PCL-g-P(DMAEMA)1, PCL-g-P(DMAEMA)2, and PCL-g-P(DMAEMA)3 surfaces, their corresponding [N]/[C] ratios (or GY values) are about 0.062 (or 0.66 μg/ cm 2 ), 0.083 (or 3.7 μg/cm2 ), and 0.096 (or 7.7 μg/cm 2 ), respectively (Figure 4 and Table 1). The above results were consistent with the fact that the increased GY of the P(DMAEMA) brushes from longer ATRP time would produce a more complete surface coverage. The average static water contact angles of the PCL-g-P(DMAEMA)3 surfaces decreased to 43° from 82° (for the starting PCL-Br surface) (Table 1), indicating that P(DMAEMA) has been successfully graft copolymerized onto the PCL-Br surface via surface-initiated ATRP.
The molecular weight and molecular weight distribution of the surface-grafted polymer cannot be determined with sufficient accuracy without the cleavage of the grafted chains. It is very difficult, if not impossible, to precisely cleave the grafted P(DMAEMA) chains from the PCL chains. Gelatin Immobilization. Gelatin has been widely used in tissue engineering, because of its excellent cell adhesion, biodegradability, and superior biocompatibility in tissue replacement and wound healing.13,16,20,22,23 It has been demonstrated that gelatin immobilization on biomaterial surfaces can improve celladhesion properties.15,20,23 The isoelectric point of ampholytic gelatin is about 4.7.18 When pH > 4.7, the net charge of gelatin is negative, and the electrostatic interaction will occur between cationic species and carboxylate groups of gelatin. It was reported that gelatin can form polyelectrolyte complexes with polycations, such as chitosan with a pKa of 6.5.18,22,23 P(DMAEMA) is a cationic polymer and its pKa is 7.07.3.16,17 Similar to chitosan, P(DMAEMA) can also form stable polyelectrolyte complexes with gelatin. In this work, the P(DMAEMA) brushes were used for immobilization of gelatin via electrostatic interaction for improving the cell-adhesion properties of the PCL surfaces. 1846
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Figure 5. AFM height and phase images of the (a,a0 ) pristine PCL, (b,b0 ) PCL-Br, (c,c0 ) PCL-g-P(DMAEMA)2, and (d,d0 ) PCL-g-P(DMAEMA)2/ Gelatin surfaces. 1847
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Figure 6. Optical images of HEK293 cells cultured for 24 h on the (a) pristine PCL, (b) PCL-g-P(DMAEMA)1, (c) PCL-g-P(DMAEMA)2, (d) PCL-gP(DMAEMA)1/Gelatin, (e) PCL-g-P(DMAEMA)2/Gelatin, and (f) PCL-g-P(DMAEMA)3/Gelatin surfaces.
The PCL-g-P(DMAEMA) surfaces were immersed into gelatin solution (pH 7.4) at 37 °C for 48 h to produce the PCL-gP(DMAEMA)/Gelatin surface. After the vigorous extraction of the reversibly and loosely bound gelatin, the C 1s core-level spectra of the PCL-g-P(DMAEMA)/Gelatin surfaces are shown in Figure 4a0 ,b0 ,c0 . In comparison with the PCL-g-P(DMAEMA) surfaces (Figure 4a,b, c, the C 1s core-level spectra of the PCL-g-P(DMAEMA)/ Gelatin surfaces possess an additional peak at about 287.4 eV, attributable to the OdCN species, 21 which is associated with the peptide bonds in gelatin. The amount of grafted P(DMAEMA) played a dominant role in the immobilization of gelatin. With the increase in the amount of the grafted P(DMAEMA) layer, the concentration of complexed gelatin is expected to increase. For the PCL-g-P(DMAEMA)1/Gelatin, PCL-g-P(DMAEMA)2/Gelatin and PCL-g-P(DMAEMA)3/ Gelatin surfaces, the [N]/[C] ratios are about 0.145, 0.162, and 0.175, respectively (Figure 4). Their corresponding immobilized amounts of gelatin are 0.7, 3.2, and 6.1 μg/cm2, respectively. In addition, the water contact angles of the PCL-gP(DMAEMA)/Gelatin surfaces were also summarized in Table 1. The changes in surface topography of the PCL film after modification were investigated by atomic force microscopy (AFM). Figure 5 shows the representative AFM height and phase images of the (a,a0 ) pristine PCL, (b,b0 ) PCL-Br, (c,c0 )
Figure 7. Relative cell-adhesion density of HEK293 cells cultured for 24 h on the pristine PCL, PCL-g-P(DMAEMA), and PCL-g-P(DMAEMA)/Gelatin surfaces, where the cell density cultured for 24 h on the pristine PCL was used as the reference. Data are presented as means ( standard deviation (n = 3).
PCL-g-P(DMAEMA)2, and (d,d0 ) PCL-g-P(DMAEMA)2/ Gelatin surfaces. The freshly prepared PCL film surface is rather uniform with a root-mean-square surface roughness value (Ra) of about 25.9 nm. After modification, the Ra values of the PCL-Br and PCL-g-P(DMAEMA)2 surfaces were 27.4 and 26.1 nm, respectively. No obvious changes in the surface roughness indicated that the ATRP initiator immobilization and subsequent 1848
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Figure 8. Representative images of pEGFP-positive HEK293 cells after 48 h post transfection with PEI/pEGFP complexes at the optimal N/P ratio of 10 on the (a) pristine PCL, (b,b0 ) PCL-g-P(DMAEMA)1/Gelatin, (c,c0 ) PCL-g-P(DMAEMA)2/Gelatin, and (d) PCL-g-P(DMAEMA)3/Gelatin surfaces.
Figure 9. Gene transfection efficiency of PEI/pEGFP complexes at the optimal N/P ratio of 10 in HEK293 cell lines on the pristine PCL, PCLg-P(DMAEMA), and PCL-g-P(DMAEMA)/Gelatin surfaces. Data are presented as means ( standard deviation (n = 3).
graft polymerization have proceeded uniformly on the PCL surfaces. After complexing with gelatin, the PCL-gP(DMAEMA)2/Gelatin surface became much rougher and the Ra value increased to about 50.9 nm. The substantial increase in surface roughness was caused by the formation of the rod-like P(DMAEMA)/gelatin complexes (as shown in Figures 5(d,d0 )).
Cell Adhesion. The cell-adhesion property of the functionalized PCL film surfaces was evaluated by culturing HEK293 cells. After 24 h of incubation, the surfaces were washed twice with the PBS solution to remove the dead and loosely attached cells. Figure 6 shows the representative optical images of PI-stained HEK293 cells cultured on the pristine PCL, PCL-g-P(DMAEMA), and PCL-g-P(DMAEMA)/Gelatin surfaces. The cells adhered and grew to some extent on the pristine PCL film surfaces. In comparison with the gelatin-immobilized PCL surfaces, the pristine PCL surfaces exhibited very poor cell attachment, due to its poor ability to interact with cells.10 After grafting P(DMAEMA) brushes, almost no attached cells were observed on the PCL-g-P(DMAEMA) surface, probably because the cationic properties of P(DMAEMA) are unfavorable to cell adhesion. It was reported that the cationic P(DMAEMA) exhibits some cytotoxicity effects on cells.24 After the gelatin immobilization, the attached cells on the PCL-g-P(DMAEMA)/ Gelatin surfaces were enhanced substantially, arising from the bioactive components of gelatin.15,20 As mentioned above, with the increase in the amount of the grafted P(DMAEMA) layer, the concentration of complexed gelatin also increased (Table 1). The higher content of the immobilized gelatin produced a higher surface coverage. The cell proliferation on the PCL-g-P(DMAEMA)/Gelatin surface should have a positive correlation with the proportion of immobilized gelatin. Unexpectedly, 1849
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Bioconjugate Chemistry among the PCL-g-P(DMAEMA)/Gelatin surfaces, the PCL-gP(DMAEMA)2/Gelatin surface (with 3.2 μg/cm2 of the immobilized gelatin) exhibited the highest level of cell attachments. The density of cell attachment on the PCL-g-P(DMAEMA)3/ Gelatin surface (with 6.1 μg/cm2 of the immobilized gelatin) decreased substantially, even lower than that on the PCL-gP(DMAEMA)1/Gelatin surface (with 0.7 μg/cm2 of the immobilized gelatin). In comparison with the roughness (Ra = 50.9 nm) of the PCL-g-P(DMAEMA)2/Gelatin surface, the PCL-g-P(DMAEMA)3/Gelatin surface was much rougher and its Ra value was about 81.2 nm. With such a rough surface and the excess amount of grafted P(DMAEMA), it is very difficult for the complexed gelatin on the PCL-g-P(DMAEMA)3/Gelatin surface (with about 6.1 μg/cm2 of grafted P(DMAEMA), Table 1) to produce efficient surface coverage. In addition, zeta potential measurements of cut segments of the PCL-g-P(DMAEMA)3/ Gelatin films confirmed that the PCL-g-P(DMAEMA)3/Gelatin film surface still possessed relatively strong positive surface charges. The above results indicated that more intact cationic groups of P(DMAEMA) on PCL-g-P(DMAEMA)3/Gelatin film surface remain and hamper the cell adhesion. The relative cell-adhesion density on the different functionalized PCL film surfaces was also investigated in Figure 7. The PCL and PCL-g-P(DMAEMA) surfaces are unfavorable to cell attachment and growth. The surfaces of the gelatin-functionalized PCL films are favorable to cell growth and survival, and the PCL-gP(DMAEMA)2/Gelatin surface with suitable graft amount of P(DMAEMA) is more favorable to cell attachment. These results are consistent with those of Figure 6. The above results indicated that the cell-adhesion process on the functionalized PCL surface can be controlled by adjusting the ratio of P(DMAEMA)/gelatin. Gene Transfection. Successful gene transfection on a tissue scaffold could enable the localized production of therapeutic drugs to facilitate tissue repair and regeneration.11,12 Thus, it is very meaningful to investigate the gene transfection in the immobilized cells on the functionalized PCL films. In this work, the subsequent gene transfection in the immobilized HEK 293 cells was investigated using the branched PEI(25KDa)/pEGFPC1 complexes in the presence of serum at the optimal N/P ratio of 10.24 Figure 8 shows the typical photos of pEGFP-positive HEK293 cells on (a) pristine PCL, (b,b0 ) PCL-g-P(DMAEMA)1/ Gelatin, (c,c0 ) PCL-g-P(DMAEMA)2/Gelatin, and (d) PCL-gP(DMAEMA)3/Gelatin surfaces, which were taken at 48 h after transfection. The densities of the transfected cells on the functionalized surfaces corresponded to those of the immobilized cells on their corresponding surfaces as shown in Figure 6. Almost no transfected cells were observed on the pristine PCL film. The PCL-g-P(DMAEMA)2/Gelatin surface exhibited the densest transfected cells, while only a few transfected cells scattered on the PCL-g-P(DMAEMA)3/Gelatin surface. The transfection efficiency (as reflected by the percentage of the EGFP positive cells) on the functionalized PCL surface was qualitatively determined using fluorescence-activated cell sorting analysis (FACS) (as shown in Figure 9). The percentages of the EGFP positive cells mediated by the PEI(25KDa)/ pEGFP-C1 complexes (at the N/P ratio of 10) on the PCL-gP(DMAEMA)1/Gelatin, PCL-g-P(DMAEMA)2/Gelatin, and PCL-g-P(DMAEMA)3/Gelatin surfaces were 19.8 ((0.6), 35.5 ((0.85), and 9.4 ((0.3)%, respectively. The transfection efficiency on the pristine PCL was only 0.7 ((0.2)%. The ctytotoxicity induced by the PEI(25KDa)/pEGFP complexes was dependent on the cell-adhesion density on the different
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functionalized PCL film surfaces. After the transfection, the cell viabilities on the PCL-g-P(DMAEMA)1/Gelatin, PCL-g-P(DMAEMA)2/Gelatin, and PCL-g-P(DMAEMA)3/Gelatin surfaces were 61%, 75%, and 40%, respectively. The higher cytotoxicity on the functionalized film surfaces with lower celladhesion density may result in a reduction in the transfection efficiency.24 The transfection efficiency on the control tissue culture polystyrene (TCPS) surface without the PCL films was 44.1((2.2)%. After the addition of the PEI(25KDa)/pEGFP complexes, the cell viability on the TCPS surface was 82%. The above results indicated that the density of the immobilized cells on the surfaces played a dominated role in the transfection efficiency of the PEI(25KDa)/pEGFP-C1 complexes, which is of crucial importance in guiding the design of multifunctional tissue scaffolds.
’ CONCLUSIONS The PCL film surfaces were successfully modified via surfaceinitiated ATRP of DMAEMA, followed by the conjugation of gelatin on the P(DMAEMA) brushes via electrostatic interaction for enhancing the cell adhesion and gene transfection properties. Kinetics study revealed an approximately linear increase in graft yield of the functional P(DMAEMA) brushes with polymerization time. The amount of complexed gelatin increases as that of the grafted P(DMAEMA) layer. The cell-adhesion process on the functionalized PCL surface can be controlled by adjusting the ratio of P(DMAEMA)/gelatin. More interestingly, the gene transfection efficiency on the immobilized cells is dependent on the density of the immobilized cells on the functionalized PCL film. With the inherent advantages of the good cell-adhesive nature of gelatin and the subsequent efficient gene transfection on the dense immobilized cells, the functionalized PCL films are potentially useful for the design of multifunctional tissue scaffolds. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (FJ Xu); majie2004@yahoo. com.cn (J Ma). Author Contributions †
Both authors contributed equally to this work.
’ ACKNOWLEDGMENT This work was supported in part by National Natural Science Foundation of China (grant numbers 50903007 and 21074007), Fok Ying Tung Education Foundation (Project no. 121048), Program for New Century Excellent Talents in University (NCET-10-0203), National Novel Drug Development Foundation of China (Grant no. 2009ZX09303-008), National Basic Research Program of China (Grant no. 2011CB911004), and Beijing Natural Science Foundation of China (Grant no. 7092086). ’ REFERENCES (1) Kweon, H. Y., Yoo, M. K., Park, I. Y., Kim, T. H., Lee, H. C., Lee, H. S., Oh, J. S., Akaike, T., and Cho, C. S. (2003) A novel degradable polycaprolactone networks for tissue engineering. Biomaterials 24, 801– 808. 1850
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