- Email: [email protected]
Hydrophobic modification of polymethyl methacrylate as intraocular lenses material to improve the cytocompatibility
Journal of Colloid and Interface Science 431 (2014) 1–7
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Hydrophobic modification of polymethyl methacrylate as intraocular lenses material to improve the cytocompatibility Bailiang Wang a,b, Quankui Lin a,b,⇑, Chenghui Shen b, Junmei Tang a, Yuemei Han a, Hao Chen a,b,⇑ a b
School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China Wenzhou Institute of Biomaterials and Engineering, Wenzhou 32500, China
a r t i c l e
i n f o
Article history: Received 20 March 2014 Accepted 27 May 2014 Available online 13 June 2014 Keywords: Posterior capsule opacification Poly (methyl methacrylate) Intraocular lenses Polyhedral oligomeric silsesquioxane
a b s t r a c t The development of posterior capsule opacification (PCO) after intraocular lenses (IOL) implantation for dealing with cataract is mainly due to the severe loss of the human lens epithelial cells (HLECs) during surgery contact. A novel poly (hedral oligomeric silsesquioxane-co-methyl methacrylate) copolymer (allyl POSS–PMMA) was synthesized by free radical polymerization method to promote the adhesion of HLECs. FT-IR and 1H NMR measurements indicated the existence of POSS cage in the product, which demonstrated the successful synthesis of allyl POSS–PMMA copolymer. Effect of allyl POSS in the hybrids on crystal structure, surface wettability and morphology, optical transmission, thermodynamic properties and cytocompatibility was investigated in detail. X-ray diffraction peaks at 2h11° and 12° indicated that POSS molecules had aggregated and crystallized. Thermogravimetric analysis-differential scanning calorimeter and optical transmission measurements confirmed that the allyl POSS–PMMA copolymer had high glass transition temperatures (more than 100 °C) and good transparency. The hydrophilicity and morphology of PMMA and copolymers surfaces were characterized by static water contact angle and atomic force microscopy. The results revealed that the surface of the allyl POSS–PMMA copolymer displayed higher hydrophobicity and higher roughness than that of pure PMMA. The surface biocompatibility was evaluated by morphology and activity measurement with HLECs in vitro. The results verified that the surface of allyl POSS–PMMA copolymer films had more HLECs adhesion and better spreading morphology than that of PMMA film. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Cataract surgery has increased rapidly in recent decades. Phacoemulsification combined with intraocular lenses (IOL) implantation is the first choice for cataract treatment on account of small incision, quick recovery and better postoperative vision [1–3]. However, posterior capsule opacification (PCO) is a common complication after cataract surgery caused by the immune response and residual human lens epithelial cells (HLECs) on the posterior capsule [4]. Wound healing promotes residual HLECs to proliferate, differentiate, and to deposit extracellular matrix, via autocrine and paracrine cell signaling. Although PCO has been extensively studied, there is no unified mechanism to explain the cause. Most current studies hypothesize that a multicellular secondary membrane results from migration and fibrosis of residual HLECs on the posterior capsule, ⇑ Corresponding authors at: Wenzhou Institute of Biomaterials and Engineering, Wenzhou 32500, China. Fax: +86 577 88067962. E-mail addresses: [email protected] (Q. Lin), [email protected] (H. Chen). http://dx.doi.org/10.1016/j.jcis.2014.05.056 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
forming elschnig pearls [5]. Others suggest that a single layer of residual anterior capsule epithelial cells migrate onto the posterior capsule and undergo metaplasia into myofibroblasts, pulling the posterior capsule into many tiny folds. Both mechanisms can contribute to the development of PCO [6]. In recent years, several reports have focused on how to prevent PCO. In addition to the position of the capsulorhexis, IOL material and optic design are important factors in the development of PCO [7–9]. The effect of IOL on PCO has been explained by various suppositions such as the separation of the posterior capsule from the anterior capsule, stretching of the capsule, compression, no space/no cells and adhesiveness of the IOL material. Out of all of the commercial IOLs, hydrophobic acrylic IOL especially poly (methyl methacrylate) (PMMA) has played an important role in cataract surgery soon after its introduction in the mid-1990s [10,11]. PMMA is the most common commercially available IOL material and is known for long-term stability. It is relatively inexpensive, inert and is well tolerated in the eye with minimal inflammatory reaction. PMMA IOL has good light transmission properties which can transmit a broad spectrum of light including near-ultraviolet
2
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
light [12]. Unfortunately, the surgery contact can cause considerable HLECs loss between the comparatively hard PMMA IOL surface and the corneal endothelium. Based on the sandwich theory [13– 15] of PCO, the rapid epithelialization of IOL that forms a cell monolayer between IOL and posterior capsule can fill up the space and finally reduce the occurrence of PCO. The surface properties of a polymer can be modified in order to ensure that it will be better adapted to its final use. Basically, the surface energy of the polymer (hydrophilic vs. hydrophobic nature) can be modified according to two general methods: surface treatment and bulk modification. The surface treatment methods of PMMA mainly include plasma treatment [16], nanoparticles doping [17] and grafting of biological macromolecules [18–20]. The plasma treatment used as a surface modification method is simple, effective and without safety issues. Using plasma discharge, hydroxyl, carboxyl or other hydrophilic functional groups can be introduced onto the surface of intraocular lenses to improve its biocompatibility. Titanium dioxide nanoparticles have been used to modify IOL to enhance its biocompatibility [17]. Heparin surface modification (HSM) decreases adhesion of cells and inflammations after cataract surgery [18]. However, a recent study shows the ratio of PCO is high using HSM IOL. Recently, a 2-methacryloyloxyethyl phosphorylchoine (MPC) coating was produced, which decreased adhesion of platelet, macrophage, lens epithelial cells and bacteria [19]. Although plasma treatment method can alter the surface wetting properties of IOL, the hydrophilic performance may lose in a short time. Metal oxide nanoparticles and biomolecules modified intraocular lenses always have color and unstable defects. Comparatively, bulk modification is a stable, effective and controllable IOL modified method. Polyhedral oligomeric silsesquioxane (POSS) is a novel cage-like structure of the organic–inorganic hybrid molecules [21–23]. The main structure of POSS consists of two parts: a cage-like inorganic core based on SiAOASi bonds and the shell composed of eight surrounded organic groups, which may be designed according to needs. The inner diameter of POSS is about 0.53 nm and the outer diameter is generally between 1 nm and 3 nm due to different organic functional groups. POSS has regular structure, good biocompatibility, small scale and large surface area, which make POSS as one of the most potential next generation biomaterials. POSS macromers with different shells have been doped into polystyrene [24], PMMA [25,26], polyurethane [27], polyethylene [28], ethylene–propylene [29], etc. by the way of copolymerization with other polymer monomers to change its mechanic, thermodynamic, surface or biological properties. Previous study [25] found that POSS and polymer hybrid is capable of forming a colorless transparent material, which does not affect the light transmittance. The cytotoxicity of POSS was also investigated and it was found that the toxicity of POSS is very low, almost non-toxic. Therefore, POSS nanomaterial is more suitable than other materials for ophthalmology biological repair alternatives. However, there is almost no reference about the studies on POSS used for IOL modification. In order to improve the biocompatibility of PMMA used as IOL material and achieve the rapid epithelialization, surface characteristics of PMMA can be changed through bulk modification. In this work, polymer of allyl POSS–PMMA was prepared using radical random copolymerization method. A schematic of the synthesis procedure is presented in Scheme 1. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of allyl POSS–PMMA copolymer and PMMA were measured by gel permeation chromatography (GPC). The effect of POSS on the crystallization, thermodynamic properties, optical performance and surface properties of allyl POSS–PMMA were studied in detail with various techniques. Furthermore, cell viability assay was performed to determine biocompatibility of the allyl POSS–PMMA copolymer with HLECs by fluorescein diacetate (FDA) and Cell Counting Kit-8 (CCK-8) methods.
2. Materials and methods 2.1. Materials and reagents Isobutyl (allyl)-POSS(R), [(allyl)(isobutyl)7Si8O12] (allyl POSS) from Hybrid Plastics Co. and methyl methacrylate (MMA), azobisisobutyronitrile (AIBN), ethyl acetate, ethanol and tetrahydrofuran (THF) from Aldrich were used as received. 2.2. Material surface preparation and characterization 2.2.1. Synthesis of allyl POSS–PMMA copolymers Allyl POSS–PMMA copolymers containing 0.01 or 0.02 weight of the allyl POSS monomers have been synthesized by free-radical polymerisation. The typical synthesis process (0.02 allyl POSS– PMMA) is described as follows: in a 50 mL round bottom flask, allyl POSS (0.34 g, 0.40 mmol), MMA (2.0 g, 20.0 mmol) and AIBN (0.025 g, 0.15 mmol) were dissolved in ethyl acetate (16 mL) and THF (4 mL) under a nitrogen atmosphere. The mixture was heated to 60 °C under constant magnetic stirring to initiate the polymerisation reaction, and the polymerisation was then carried out at the elevated temperature for 24 h. After the reaction, the solution was dropped into excess ethanol to precipitate the polymer. The polymer was then purified via three dissolving/precipitating cycles, and finally dried at 30 °C in vacuum for 24 h. 2.2.2. Preparation of material surfaces The material surfaces of PMMA and allyl POSS–PMMA were spin-coated onto many kinds of substrates including glass slide, silicon wafer, PET sheet and quartz plate (1 2 cm2) from ethyl acetate (0.5% (w/w) at 1500 rpm for 60 s). The coatings were dried at 25 °C for 24 h and for 12 h under vacuum at 30 °C. Glass slide, silicon wafer and quartz plate used for coating preparation were cleaned in ‘‘piranha’’ (7:3 (v/v) H2SO4/H2O2) for 1 min and water for 10 min respectively, and then dried with N2. 2.2.3. Characterization of the polymers and material surfaces 2.2.3.1. Characterization of the polymers. Molecular weights and distributions of all polymer samples were characterized by GPC performed in THF (1.0 mL/min). Calibration was carried out using a series of near-monodisperse polystyrene standards. 1H NMR was measured with a Bruker 400 NMR spectrometer at 25 °C, using deuterio-chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as the internal standard. Fourier transform infrared spectra (FT-IR) were measured on a FT-IR spectrometry (Bruker Optics). X-ray diffraction (XRD) was performed on a powder diffractometer (Philips 1140/90) using Cu radiation 1.54 Å. The samples were analyzed at room temperature over a 2h range of 5–50° with sampling intervals of 0.04°. 2.2.3.2. Characterization of material surfaces. 2.2.3.2.1. Surface morphology. Surface morphology was measured by atomic force microscope (AFM, SPA 400, Seiko instrument Inc.). AFM images were performed in the tapping mode under ambient conditions using a commercial scanning probe microscope, equipped with a silicon cantilever, nanosensors, typical spring constant 40 N m 1. 2.2.3.2.2. Surface wettability. Surface wettability of the films was } DSA10-MK2). The sessile measured by Drop Shape Analysis (KRUSS, dropping method was used to detect surface of the film with different times after the ultrapure water droplet contacted the film. The contact angle formed between the sample surface and droplet was measured using built-in microscope and software provided by manufacturer. All the measurements were performed at least in triplicate and the data were presented as mean ± standard deviation.
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
3
Scheme 1. Synthetic route for allyl POSS–PMMA copolymer.
2.2.3.2.3. Optical transmission. The optical transmission (OT) of PMMA and allyl POSS–PMMA surfaces was performed on a quartz plate followed by measurement of its transmission using UV–Vis spectrophotometer from 350 to 800 nm. The quartz plate was used as reference. Three film samples were used for measurement of OT. And the results were expressed as mean value. 2.2.3.2.4. Thermodynamic properties. Thermogravimetric analysis (TGA) was performed with an instrument from Thermal Analysis Incorporation (TA-TGA 2050) at a heating rate of 10 °C /min under nitrogen atmosphere from room temperature to 550 °C. The phase transformation behavior of the experimental specimens was characterized by differential scanning calorimetry (DSC), using a Perkin–Elmer Diamond calorimeter with a heating and cooling rate of 20 K min 1.
2.2.4.3. Cell morphology. FDA (Sigma) is an indicator of membrane integrity and cytoplasmic esterase activity. The cell monolayers on different surfaces were stained with FDA for fluorescence microscope investigation (Zeiss, Germany) at 10 magnification in fluorescein filter, 488 nm excitation. Stock solutions were prepared by dissolving 5.0 mg/mL FDA in acetone. The working solution was freshly prepared by adding 5.0 lL of FDA stock solution into 5.0 mL of PBS. FDA solution (20 lL) was added into each well of a 96-well plate and incubated for 5 min. The sheets were then washed twice with PBS and placed on a glass slide for fluorescence microscope examination. The 488 nm wavelength of the laser was used to excite the dye. Cells incubated into wells that did not contain films were used as controls.
2.2.4. Cytotoxicity assays 2.2.4.1. Cell cultivation. The HLECs (HLE-B3, ATCC Number CRL11421™) were grown in DMEM/F12 (1:1) mixed medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL streptomycin in a 5% CO2 incubator at 37 °C. Confluent cells were digested using 0.25% trypsin–0.02% EDTA, followed by centrifugation (1000 g for 3 min) to harvest the cells. Subsequently, the single cell suspension was used for cell number calculation using haemacytometer. After confluence, cells were digested and resuspended for cultivation on the materials. The HLECs were seeded onto the specimens at a density of 1.0 104 cells per sample by using 96-well tissue culture plate as the holder, cultivation was conducted for 24 h. Then, FDA and CCK-8 assays were used for the viability and morphology studies of cells grown on the resulting films.
3. Results and discussion
2.2.4.2. Cell viability. CCK-8 (Beyotime, China) assay was employed in this experiment to quantitatively evaluate the cell viability. After HLECs were inoculated on the film coated dishes for 24 h, the original medium was replaced by 100 lL 10% FBS DMEM/F12 (1:1) mixed medium contains 10 lL CCK-8. It was incubated at 37 °C for 2 h to form water dissoluble formazan. Then 100 lL of the above formazan solution were taken from each sample and added to one well of a 96-well plate, six parallel replicates were prepared. The absorbance at 450 nm (calibrated wave) was determined using a microplate reader (Multiskan MK33, Thermo electron corporation, China). Tissue culture plates (TCPS) without any film were used as a control.
3.1. Synthesis of allyl POSS–PMMA copolymers The allyl POSS–PMMA copolymer was synthesized via free radical polymerization using AIBN as the initiator, allyl POSS and MMA as monomers. The successful synthesis of copolymers was proved by 1H NMR. Fig. 1. is the typical 1H NMR spectra of 0.02 allyl POSS–PMMA copolymer recorded in CDCl3 with the relevant signals labeled. The characteristic resonance signals at 0.60 ppm, 0.91 ppm, 1.81 ppm, and 1.85 ppm were attributed to allyl POSS, while signals at 0.85 ppm, 1.03 ppm, 1.25 ppm, 1.60 ppm and 3.60 ppm belonged to PMMA. The Mn/Mw and polydispersity (PDI) of PMMA and allyl POSS– PMMA copolymers were calculated from GPC using THF as eluent. The characterization of PMMA and the copolymer is displayed in Table 1. GPC measurement indicated a unimodal molecular weight distribution for both the resultant copolymers and pure PMMA. The Mw/Mn for the 0.01 allyl POSS–PMMA copolymer was found to be 480,500/206,300 g/mol with a PDI of 2.38, typical for copolymers synthesized by free radical polymerization. The Mw/Mn for the 0.02 allyl POSS–PMMA copolymer was found to be 562,000/ 220,600 g/mol with a PDI of 2.49 (see Fig. 2.). For the pure PMMA, the Mw/Mn and PDI were found to be 448,600/210,400 g/mol and 2.13, respectively. Changes in chemical properties of PMMA to allyl POSS–PMMA copolymer due to introduce of POSS cage were examined using
4
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
Fig. 1. 1H-NMR spectrum of 0.02 allyl POSS–PMMA copolymer.
Table 1 Copolymerization of MMA and allyl POSS with free radical polymerization. Polymer Mna/g mol Mwb/g mol PDIc
1 1
PMMA
0.01 allyl POSS + PMMA
0.02 allyl POSS + PMMA
210,400 448,600 2.13
206,300 490,500 2.38
220,600 562,000 2.49
a The number-average molecular weight (Mn) were determined by gel permeation chromatography (GPC) performed in THF (1.0 ml/min). b Weight-average molecular meight (Mw) were determined by gel permeation chromatography (GPC) performed in THF (1.0 ml/min). c The molecular weight distributions were calculated as Mw/Mn.
FT-IR spectral analysis. The FT-IR spectrum of the allyl POSS– PMMA copolymer showed combined features for PMMA and allyl POSS (see Fig. 3). There were three strong vibration bands at 1637.0 cm 1, 1728.5 cm 1 and 1148.3 cm 1, attributable to C@O and CAOAC stretching vibrations from PMMA. The strong SiAOASi stretching vibration at 1115.2 cm 1 from the POSS cage indicated the existence of allyl POSS in copolymers [30], which demonstrated the successful synthesis of allyl POSS–PMMA copolymer. The X-ray scattering profiles of PMMA and allyl POSS–PMMA copolymer are shown in Fig. 4. The shape of the most intense peak reflects the ordered packing of polymer chains. The pure PMMA exhibited reflections 2h’s at 14° which is amorphous arrangement of polymer segment. For the 0.01 allyl POSS–PMMA copolymer, there were peaks 2h at 11° and 12° indicating regular arrangement of allyl POSS molecules. As the allyl POSS content increased to 0.02, the reflections (2h = ca. 11° and 12°) from allyl POSS component increased in intensity and sharpness while the new reflections at 8° and 9° were stronger and sharper. From this observation, it was confirmed that the allyl POSS cages formed the aggregation and crystalline domain.
Fig. 2. GPC chromatogram of 0.02 allyl POSS–PMMA copolymer.
3.2. Characterization of the material surfaces Fig. 5 shows the water contact angle (WCA) for the PMMA and allyl POSS–PMMA copolymer films with the extension of time from 5 s to 1440 s. In all cases, WCA was gradually decreased during the
Fig. 3. FT-IR spectra of PMMA and 0.02 allyl POSS–PMMA copolymer.
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
5
Fig. 4. X-ray diffraction patterns of PMMA and allyl POSS–PMMA copolymers. Fig. 7. Optical transmittance of the PMMA and allyl POSS–PMMA copolymer films.
Fig. 5. WCAs of the PMMA and allyl POSS–PMMA copolymer surfaces with the change of time. Fig. 8. TGA derivative plots of PMMA and allyl POSS–PMMA copolymers.
Fig. 9. DSC thermograms of PMMA and allyl POSS–PMMA copolymers.
Fig. 6. AFM images of films of (a) PMMA, (b) 0.01 allyl POSS–PMMA and (c) 0.02 allyl POSS–PMMA.
initial 900 s and reached plateau after that time. PMMA films showed hydrophobic property with WCAs of 94.7 ± 6.7° at 10 s and 74.5 ± 7.2° at 1440 s, while films consisting of allyl POSS had higher hydrophobicity with higher WCA values than PMMA film. The WCAs of allyl POSS–PMMA copolymer films (0.01 and 0.02)
6
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
Fig. 10. The cell viability assay of HLECs cultured on the surfaces of PMMA and allyl POSS–PMMA copolymers for 24 h. The absorbance of the diluted cell counting kit solution has been deducted from each data point and the statistical significance is indicated by different letters (p < 0.05).
were 101.8 ± 4.1° and 105.7 ± 3.0° at 10 s and 86.4 ± 3.8° and 88.2 ± 4.1° at 1440 s, respectively. The WCAs of the copolymers also showed that with the increase of allyl POSS from 0.01 to 0.02, the hydrophobicity slightly increased [25,31]. To further understand possible effect of the material composition on the surface morphology, we used AFM to study the morphology of spin-coated films. Photo images given in Fig. 6 showed the 3D images of the copolymers and pure PMMA films. It can be observed that the allyl POSS–PMMA copolymer films had a slightly higher roughness than pure PMMA film with RMS roughness of 2.96 ± 0.46 nm. As the adding of allyl POSS changing from 0.01 to 0.02, the RMS roughness of the films increased from 4.60 ± 0.52 nm to 5.84 ± 0.56 nm. The increase of roughness of the surface contributed to the hydrophobicity increase of copolymer films [32,33].
The transmittance of PMMA is also of great importance in its actual biomedical applications, especially used as intraocular lenses. So the transmittance of allyl POSS–PMMA copolymer films was also examined in this research, as shown in Fig. 7. It illustrated that the transmittance of pristine PMMA in visible region (400– 700 nm) was very excellent, with light transmission higher than 93%. However, the complex of allyl POSS in PMMA resulted in the decrease of the light transmission. The transmittances of 0.01 and 0.02 allyl POSS–PMMA copolymer films were respectively 91–93% and 90–92% in visible region. So the amount of doping allyl POSS should be controlled so as to keep transmittance of allyl POSS–PMMA copolymer films in a reasonable range. The thermal analysis was carried out by TGA and DSC. As seen in Fig. 8, the pure PMMA shows three-step thermal decomposition behaviors (160–260 °C, 260–310 °C and 310–400 °C), while the amphiphilic allyl POSS–PMMA hybrids exhibit clearly two-step thermal decomposition behaviors (160–260 °C and 260–400 °C), suggesting the altered thermal decomposition due to the incorporation of allyl POSS macromolecules [34]. The glass transition temperature (Tg) was taken as the inflection point of the heat flow of the second heating run. Fig. 9 showed the DSC heating traces of neat PMMA and allyl POSS–PMMA copolymer at 20 °C/min from the amorphous state. Neat PMMA exhibits a Tg around 115.7 °C. Incorporation of POSS comonomers into PMMA caused a slight decrease of Tg, while maintaining thermal stability. The Tg of 0.01 and 0.02 allyl POSS–PMMA copolymer were 111.5 °C and 102.3 °C, respectively. The slight decrease in Tg with an increase of allyl POSS content indicated that incorporation of allyl POSS macromolecule as a pendent group onto PMMA backbone facilitated the movement of polymer chains and resulted in crystallites with lower thermal stability [35]. 3.3. Cytotoxicity of the material surfaces The growth and proliferation of the HLECs on the surfaces of PMMA and allyl POSS–PMMA copolymer films were investigated
Fig. 11. Growth and morphology of HLECs stained with FDA after 24 h of incubation on (a) TCPS, (b) pristine PMMA film, (c) 0.01 allyl POSS–PMMA copolymer film, (d) 0.02 allyl POSS–PMMA copolymer film under fluorescence microscopy (the magnification is 10).
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
to characterize the cell compatibility of the materials. Fig. 10 showed quantitative assessment of the cytotoxicity by CCK-8 assay over a period of 24 h. The results were the average of three replicate experiments. It was found that the cell viability of PMMA and allyl POSS–PMMA copolymer films was much lower than that of TCPS. It also could be seen that PMMA film had the lowest cell viability compared with TCPS and allyl POSS–PMMA copolymers. With the complex of allyl POSS, the cell viability increased from 0.409 ± 0.098 to 0.472 ± 0.103 and 0.498 ± 0.088 for 0.01 and 0.02 allyl POSS–PMMA copolymer films respectively. Surface properties have an important impact on not only cell viability, but also cell adhesion and proliferation [36,37]. The adhered HLECs on the surface were photographed with an inverted fluorescence microscope after FDA staining, and the results are shown in Fig. 11. After 24 h incubation, it could be seen that HLECs incubated on TCPS had changed their morphology to longer and bigger structures together with an obvious increase in number, suggesting the HLECs were in the growth status (Fig. 11(a)). The HLECs cultured on the PMMA and allyl POSS–PMMA copolymer films had undergone some degree of proliferation. However, the number of cells attachment was less comparing with TCPS. Simultaneously, the surfaces of allyl POSS–PMMA copolymer films had more HLECs and better spreading morphology than that on the surface of PMMA. This was consistent with the results of CCK-8. It has been reported that the surface chemistry of a material can mediate the cellular response to the material and affect cellular function on the surface including cell adhesion, proliferation and migration [38,39]. Surface wettability was the most important factor [40]. Studies of the influence of WCA on cell attachment and spreading further showed that cells exhibited poor attachment and spreading on a materials surface with a low WCA [41]. Cell membrane with hydrophobic property prefers hydrophobic material surfaces due to hydrophobic interactions [42,43]. The WCAs of all allyl POSS–PMMA copolymer films were higher than PMMA film, which promoted HLECs attachment and spreading. The adhesion of cells to different substrates surfaces is also affected by various chemical and physicochemical factors, such as roughness. According to sandwich theory of PCO [13–15], the promotion of HLECs adhesion on IOL can form a cell monolayer between IOL and posterior capsule, fill up the space and reduce the occurrence of PCO.
4. Conclusion Allyl POSS–PMMA copolymer was synthesized by radical polymerization and made into film as IOL material. FT-IR, XRD and 1H NMR measurements indicated the successful synthesis of allyl POSS–PMMA copolymer. By incorporating of ally POSS into the PMMA main chain results in good thermodynamic properties and high transparency of PMMA-based polymeric material. Characterizations of morphology and surface hydrophility suggested that the allyl POSS–PMMA copolymer film had a higher hydrophobicity and a higher roughness. The surface of allyl POSS–PMMA films had better cellular compatibility with HLECs and better spreading morphology than pure PMMA film. As a result, the presence of allyl POSS in PMMA in some extent promoted the epithelialization of HLECs on the surface and may reduce the occurrence of PCO, which may be of great interest being used as a novel IOL material.
7
Acknowledgments Financial supports from National Natural Science Foundation of China (81271703, 51203120), Natural Science Foundation of Zhejiang Province (LQ12E03001), Medical & Health Technology Program of Zhejiang Province (2013KYA133, 2014KYA149), the International Scientific and Technological Cooperation Projects (2012DFB30020) and the Specialized Research Fund for Science and Technology Major Projects of Higher Education of China (ZD2007006) are greatly acknowledged. References [1] E.J. Hollick, D.J. Spalton, P.G. Ursell, Arch. Ophthalmol. 117 (1999) 872. [2] J.W. Cheng, R.L. Wei, J.P. Cai, G.L. Xi, H. Zhu, Y. Li, X.Y. Ma, Am. J. Ophthalmol. 143 (2007) 428. [3] N. Li, X. Chen, J. Zhang, Y. Zhou, X. Yao, L. Du, M. Wei, Y. Liu, Ophthalmology 115 (2008) 830. [4] P.J. Morgan-Warren, J.A. Smith, Clin. Ophthalmol. 7 (2013) 1661. [5] H. Matsushima, H. Iwamoto, K. Mukai, Y. Katsuki, M. Nagata, T. Senoo, Expert Rev. Med. Dev. 5 (2008) 197. [6] D.M. Saylor, D.C. Richardson, B.J. Dair, S.K. Pollack, Acta Biomater. 6 (2010) 1090. [7] S. Sacu, R. Menapace, W. Buehl, G. Rainer, O. Findl, J. Cataract Refract. Surg. 30 (2004) 1875. [8] S. Argal, J. Clin. Ophthalmol. Res. 1 (2013) 113. [9] B. Amoozgar, D. Morarescu, H. Sheardown, Coll. Surf. B 111 (2013) 15. [10] S. Hazra, H. Palui, G.K. Vemuganti, Int. J. Ophthalmol. 5 (2012) 59. [11] S.M. Shah, D.J. Spalton, J. Cataract Refract. 21 (1995) 579. 579.J. [12] O. Findl, W. Buehl, R. Menapace, S. Sacu, M. Georgopoulos, G. Rainer, Ophthalmology 112 (2005) 2004. [13] R.J. Linnola, A. Holst, J. Cataract Refract. 24 (2001) 1509. [14] R.J. Linnola, J. Cataract Refract. 23 (1997) 1539. [15] S.M. Raj, A.R. Vasavada, S.R. Johar, V.A. Vasavada, Int. J. Biomed. Sci. 3 (2007) 237. [16] R.A. D’Sa, J. Raj, M.A.S. McMahon, D.A. McDowell, G.A. Burke, B.J. Meenan, J. Colloid Interface Sci. 375 (2012) 193. [17] M.J. Yang, Z.F. Di, J.K. Lee, J. Colloid Interface Sci. 368 (2012) 603. [18] S. Kang, M.J. Kim, S.H. Park, C.K. Joo, Euro. J. Ophthalmol. 18 (2008) 377. [19] X.D. Huang, K. Yao, Z. Zhang, Y.D. Zhang, Y. Wang, J. Cataract Refract. 36 (2010) 290. [20] L. Lin, Y. Wang, X.D. Huanga, Z.K. Xu, K. Yao, Appl. Surf. Sci. 256 (2010) 7354. [21] S.H. Phillips, T.S. Haddad, S.J. Tomczak, Interface Sci. 8 (2004) 21–29. [22] R.Y. Kannan, H.J. Salacinski, P.E. Butler, Acc. Chem. Res. 38 (2005) 879. [23] R.Y. Kannan, H.J. Salacinski, M.J. Edirisinghe, Biomaterials 27 (2006) 4618. [24] X.Y. Song, H.P. Geng, Q.F. Li, Polymer 47 (2006) 3049. [25] Y. Xue, H. Wang, D. Yu, L. Fen, Chem. Commun. 42 (2009) 6418. [26] H. Xu, B. Yang, J. Wang, S. Guang, C. Li, J. Poly. Sci. Part A: Poly. Chem. 45 (2007) 5308. [27] H. Liu, S. Zheng, Macromol. Rapid Commun. 26 (2005) 196. [28] P.T. Mather, H.G. Jeon, A. Romo-Uribe, T.S. Haddad, J.D. Lichtenhan, Macromolecules 32 (1999) 1194. [29] B. Seurer, E.B. Coughlin, Polym. Prepr. 46 (2005) 738. [30] Y.L. Liu, M.C. Tseng, M.H. Fangchiang, J. Poly. Sci. Part A: Poly. Chem. 46 (2008) 5157. [31] A. Tuteja, W. Choi, M. Ma, J.M. Mabry, S.A. Mazzella, G.C. Rutledge, G.H. McKinley, R.E. Cohen, Science 318 (2007) 1618. [32] Y. Tian, L. Jiang, Nat. Mater. 12 (2013) 291. [33] K.J. Kubiaka, M.C.T. Wilsona, T.G. Mathiab, P. Carvalc, Wear 271 (2011) 523. [34] C.K. Kim, B.S. Kim, F.A. Sheikh, U.S. Lee, M.S. Khil, H.Y. Kim, Macromolecules 40 (2007) 4823. [35] Y. Yani, M.H. Lamm, Polymer 50 (2009) 1324. [36] S.S. Li, D.Y. Yang, H.Y. Tu, H.T. Deng, D. Du, A.D. Zhang, J. Colloid Interface Sci. 402 (2013) 284. [37] B. Cortese, M.O. Riehle, S. D’Amon, G. Gigli, J. Colloid Interface Sci. 394 (2013) 582. [38] Z. Zheng, F.F. Bei, H.L. Tian, G.Q. Chen, Biomaterials 26 (2005) 3537. [39] Z.W. Dai, X.H. Zou, G.Q. Chen, Biomaterials 30 (2009) 3075. [40] N.J. Hallab, K.J. Bundy, K. O’Connor, R.L. Moses, J.J. Jacobs, Tissue Eng. 7 (2001) 55. [41] L.H. Zhang, D.W.Y.S. Chen, X.L. Wang, G.W. Zhao, H.Y. Wang, C.Z. Huang, Appl. Surf. Sci. 255 (2009) 6840. [42] J.D. Mendelsohn, S.Y. Yang, J.A. Hiller, A.I. Hochbaum, M.F. Rubner, Biomacromolecules 4 (2003) 96–106. [43] B.L. Wang, J.L. Wang, D.D. Li, K.F. Ren, J. Ji, Appl. Surf. Sci. 258 (2012) 7801.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Hydrophobic modification of polymethyl methacrylate as intraocular lenses material to improve the cytocompatibility Bailiang Wang a,b, Quankui Lin a,b,⇑, Chenghui Shen b, Junmei Tang a, Yuemei Han a, Hao Chen a,b,⇑ a b
School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China Wenzhou Institute of Biomaterials and Engineering, Wenzhou 32500, China
a r t i c l e
i n f o
Article history: Received 20 March 2014 Accepted 27 May 2014 Available online 13 June 2014 Keywords: Posterior capsule opacification Poly (methyl methacrylate) Intraocular lenses Polyhedral oligomeric silsesquioxane
a b s t r a c t The development of posterior capsule opacification (PCO) after intraocular lenses (IOL) implantation for dealing with cataract is mainly due to the severe loss of the human lens epithelial cells (HLECs) during surgery contact. A novel poly (hedral oligomeric silsesquioxane-co-methyl methacrylate) copolymer (allyl POSS–PMMA) was synthesized by free radical polymerization method to promote the adhesion of HLECs. FT-IR and 1H NMR measurements indicated the existence of POSS cage in the product, which demonstrated the successful synthesis of allyl POSS–PMMA copolymer. Effect of allyl POSS in the hybrids on crystal structure, surface wettability and morphology, optical transmission, thermodynamic properties and cytocompatibility was investigated in detail. X-ray diffraction peaks at 2h11° and 12° indicated that POSS molecules had aggregated and crystallized. Thermogravimetric analysis-differential scanning calorimeter and optical transmission measurements confirmed that the allyl POSS–PMMA copolymer had high glass transition temperatures (more than 100 °C) and good transparency. The hydrophilicity and morphology of PMMA and copolymers surfaces were characterized by static water contact angle and atomic force microscopy. The results revealed that the surface of the allyl POSS–PMMA copolymer displayed higher hydrophobicity and higher roughness than that of pure PMMA. The surface biocompatibility was evaluated by morphology and activity measurement with HLECs in vitro. The results verified that the surface of allyl POSS–PMMA copolymer films had more HLECs adhesion and better spreading morphology than that of PMMA film. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Cataract surgery has increased rapidly in recent decades. Phacoemulsification combined with intraocular lenses (IOL) implantation is the first choice for cataract treatment on account of small incision, quick recovery and better postoperative vision [1–3]. However, posterior capsule opacification (PCO) is a common complication after cataract surgery caused by the immune response and residual human lens epithelial cells (HLECs) on the posterior capsule [4]. Wound healing promotes residual HLECs to proliferate, differentiate, and to deposit extracellular matrix, via autocrine and paracrine cell signaling. Although PCO has been extensively studied, there is no unified mechanism to explain the cause. Most current studies hypothesize that a multicellular secondary membrane results from migration and fibrosis of residual HLECs on the posterior capsule, ⇑ Corresponding authors at: Wenzhou Institute of Biomaterials and Engineering, Wenzhou 32500, China. Fax: +86 577 88067962. E-mail addresses: [email protected] (Q. Lin), [email protected] (H. Chen). http://dx.doi.org/10.1016/j.jcis.2014.05.056 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
forming elschnig pearls [5]. Others suggest that a single layer of residual anterior capsule epithelial cells migrate onto the posterior capsule and undergo metaplasia into myofibroblasts, pulling the posterior capsule into many tiny folds. Both mechanisms can contribute to the development of PCO [6]. In recent years, several reports have focused on how to prevent PCO. In addition to the position of the capsulorhexis, IOL material and optic design are important factors in the development of PCO [7–9]. The effect of IOL on PCO has been explained by various suppositions such as the separation of the posterior capsule from the anterior capsule, stretching of the capsule, compression, no space/no cells and adhesiveness of the IOL material. Out of all of the commercial IOLs, hydrophobic acrylic IOL especially poly (methyl methacrylate) (PMMA) has played an important role in cataract surgery soon after its introduction in the mid-1990s [10,11]. PMMA is the most common commercially available IOL material and is known for long-term stability. It is relatively inexpensive, inert and is well tolerated in the eye with minimal inflammatory reaction. PMMA IOL has good light transmission properties which can transmit a broad spectrum of light including near-ultraviolet
2
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
light [12]. Unfortunately, the surgery contact can cause considerable HLECs loss between the comparatively hard PMMA IOL surface and the corneal endothelium. Based on the sandwich theory [13– 15] of PCO, the rapid epithelialization of IOL that forms a cell monolayer between IOL and posterior capsule can fill up the space and finally reduce the occurrence of PCO. The surface properties of a polymer can be modified in order to ensure that it will be better adapted to its final use. Basically, the surface energy of the polymer (hydrophilic vs. hydrophobic nature) can be modified according to two general methods: surface treatment and bulk modification. The surface treatment methods of PMMA mainly include plasma treatment [16], nanoparticles doping [17] and grafting of biological macromolecules [18–20]. The plasma treatment used as a surface modification method is simple, effective and without safety issues. Using plasma discharge, hydroxyl, carboxyl or other hydrophilic functional groups can be introduced onto the surface of intraocular lenses to improve its biocompatibility. Titanium dioxide nanoparticles have been used to modify IOL to enhance its biocompatibility [17]. Heparin surface modification (HSM) decreases adhesion of cells and inflammations after cataract surgery [18]. However, a recent study shows the ratio of PCO is high using HSM IOL. Recently, a 2-methacryloyloxyethyl phosphorylchoine (MPC) coating was produced, which decreased adhesion of platelet, macrophage, lens epithelial cells and bacteria [19]. Although plasma treatment method can alter the surface wetting properties of IOL, the hydrophilic performance may lose in a short time. Metal oxide nanoparticles and biomolecules modified intraocular lenses always have color and unstable defects. Comparatively, bulk modification is a stable, effective and controllable IOL modified method. Polyhedral oligomeric silsesquioxane (POSS) is a novel cage-like structure of the organic–inorganic hybrid molecules [21–23]. The main structure of POSS consists of two parts: a cage-like inorganic core based on SiAOASi bonds and the shell composed of eight surrounded organic groups, which may be designed according to needs. The inner diameter of POSS is about 0.53 nm and the outer diameter is generally between 1 nm and 3 nm due to different organic functional groups. POSS has regular structure, good biocompatibility, small scale and large surface area, which make POSS as one of the most potential next generation biomaterials. POSS macromers with different shells have been doped into polystyrene [24], PMMA [25,26], polyurethane [27], polyethylene [28], ethylene–propylene [29], etc. by the way of copolymerization with other polymer monomers to change its mechanic, thermodynamic, surface or biological properties. Previous study [25] found that POSS and polymer hybrid is capable of forming a colorless transparent material, which does not affect the light transmittance. The cytotoxicity of POSS was also investigated and it was found that the toxicity of POSS is very low, almost non-toxic. Therefore, POSS nanomaterial is more suitable than other materials for ophthalmology biological repair alternatives. However, there is almost no reference about the studies on POSS used for IOL modification. In order to improve the biocompatibility of PMMA used as IOL material and achieve the rapid epithelialization, surface characteristics of PMMA can be changed through bulk modification. In this work, polymer of allyl POSS–PMMA was prepared using radical random copolymerization method. A schematic of the synthesis procedure is presented in Scheme 1. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of allyl POSS–PMMA copolymer and PMMA were measured by gel permeation chromatography (GPC). The effect of POSS on the crystallization, thermodynamic properties, optical performance and surface properties of allyl POSS–PMMA were studied in detail with various techniques. Furthermore, cell viability assay was performed to determine biocompatibility of the allyl POSS–PMMA copolymer with HLECs by fluorescein diacetate (FDA) and Cell Counting Kit-8 (CCK-8) methods.
2. Materials and methods 2.1. Materials and reagents Isobutyl (allyl)-POSS(R), [(allyl)(isobutyl)7Si8O12] (allyl POSS) from Hybrid Plastics Co. and methyl methacrylate (MMA), azobisisobutyronitrile (AIBN), ethyl acetate, ethanol and tetrahydrofuran (THF) from Aldrich were used as received. 2.2. Material surface preparation and characterization 2.2.1. Synthesis of allyl POSS–PMMA copolymers Allyl POSS–PMMA copolymers containing 0.01 or 0.02 weight of the allyl POSS monomers have been synthesized by free-radical polymerisation. The typical synthesis process (0.02 allyl POSS– PMMA) is described as follows: in a 50 mL round bottom flask, allyl POSS (0.34 g, 0.40 mmol), MMA (2.0 g, 20.0 mmol) and AIBN (0.025 g, 0.15 mmol) were dissolved in ethyl acetate (16 mL) and THF (4 mL) under a nitrogen atmosphere. The mixture was heated to 60 °C under constant magnetic stirring to initiate the polymerisation reaction, and the polymerisation was then carried out at the elevated temperature for 24 h. After the reaction, the solution was dropped into excess ethanol to precipitate the polymer. The polymer was then purified via three dissolving/precipitating cycles, and finally dried at 30 °C in vacuum for 24 h. 2.2.2. Preparation of material surfaces The material surfaces of PMMA and allyl POSS–PMMA were spin-coated onto many kinds of substrates including glass slide, silicon wafer, PET sheet and quartz plate (1 2 cm2) from ethyl acetate (0.5% (w/w) at 1500 rpm for 60 s). The coatings were dried at 25 °C for 24 h and for 12 h under vacuum at 30 °C. Glass slide, silicon wafer and quartz plate used for coating preparation were cleaned in ‘‘piranha’’ (7:3 (v/v) H2SO4/H2O2) for 1 min and water for 10 min respectively, and then dried with N2. 2.2.3. Characterization of the polymers and material surfaces 2.2.3.1. Characterization of the polymers. Molecular weights and distributions of all polymer samples were characterized by GPC performed in THF (1.0 mL/min). Calibration was carried out using a series of near-monodisperse polystyrene standards. 1H NMR was measured with a Bruker 400 NMR spectrometer at 25 °C, using deuterio-chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as the internal standard. Fourier transform infrared spectra (FT-IR) were measured on a FT-IR spectrometry (Bruker Optics). X-ray diffraction (XRD) was performed on a powder diffractometer (Philips 1140/90) using Cu radiation 1.54 Å. The samples were analyzed at room temperature over a 2h range of 5–50° with sampling intervals of 0.04°. 2.2.3.2. Characterization of material surfaces. 2.2.3.2.1. Surface morphology. Surface morphology was measured by atomic force microscope (AFM, SPA 400, Seiko instrument Inc.). AFM images were performed in the tapping mode under ambient conditions using a commercial scanning probe microscope, equipped with a silicon cantilever, nanosensors, typical spring constant 40 N m 1. 2.2.3.2.2. Surface wettability. Surface wettability of the films was } DSA10-MK2). The sessile measured by Drop Shape Analysis (KRUSS, dropping method was used to detect surface of the film with different times after the ultrapure water droplet contacted the film. The contact angle formed between the sample surface and droplet was measured using built-in microscope and software provided by manufacturer. All the measurements were performed at least in triplicate and the data were presented as mean ± standard deviation.
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
3
Scheme 1. Synthetic route for allyl POSS–PMMA copolymer.
2.2.3.2.3. Optical transmission. The optical transmission (OT) of PMMA and allyl POSS–PMMA surfaces was performed on a quartz plate followed by measurement of its transmission using UV–Vis spectrophotometer from 350 to 800 nm. The quartz plate was used as reference. Three film samples were used for measurement of OT. And the results were expressed as mean value. 2.2.3.2.4. Thermodynamic properties. Thermogravimetric analysis (TGA) was performed with an instrument from Thermal Analysis Incorporation (TA-TGA 2050) at a heating rate of 10 °C /min under nitrogen atmosphere from room temperature to 550 °C. The phase transformation behavior of the experimental specimens was characterized by differential scanning calorimetry (DSC), using a Perkin–Elmer Diamond calorimeter with a heating and cooling rate of 20 K min 1.
2.2.4.3. Cell morphology. FDA (Sigma) is an indicator of membrane integrity and cytoplasmic esterase activity. The cell monolayers on different surfaces were stained with FDA for fluorescence microscope investigation (Zeiss, Germany) at 10 magnification in fluorescein filter, 488 nm excitation. Stock solutions were prepared by dissolving 5.0 mg/mL FDA in acetone. The working solution was freshly prepared by adding 5.0 lL of FDA stock solution into 5.0 mL of PBS. FDA solution (20 lL) was added into each well of a 96-well plate and incubated for 5 min. The sheets were then washed twice with PBS and placed on a glass slide for fluorescence microscope examination. The 488 nm wavelength of the laser was used to excite the dye. Cells incubated into wells that did not contain films were used as controls.
2.2.4. Cytotoxicity assays 2.2.4.1. Cell cultivation. The HLECs (HLE-B3, ATCC Number CRL11421™) were grown in DMEM/F12 (1:1) mixed medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL streptomycin in a 5% CO2 incubator at 37 °C. Confluent cells were digested using 0.25% trypsin–0.02% EDTA, followed by centrifugation (1000 g for 3 min) to harvest the cells. Subsequently, the single cell suspension was used for cell number calculation using haemacytometer. After confluence, cells were digested and resuspended for cultivation on the materials. The HLECs were seeded onto the specimens at a density of 1.0 104 cells per sample by using 96-well tissue culture plate as the holder, cultivation was conducted for 24 h. Then, FDA and CCK-8 assays were used for the viability and morphology studies of cells grown on the resulting films.
3. Results and discussion
2.2.4.2. Cell viability. CCK-8 (Beyotime, China) assay was employed in this experiment to quantitatively evaluate the cell viability. After HLECs were inoculated on the film coated dishes for 24 h, the original medium was replaced by 100 lL 10% FBS DMEM/F12 (1:1) mixed medium contains 10 lL CCK-8. It was incubated at 37 °C for 2 h to form water dissoluble formazan. Then 100 lL of the above formazan solution were taken from each sample and added to one well of a 96-well plate, six parallel replicates were prepared. The absorbance at 450 nm (calibrated wave) was determined using a microplate reader (Multiskan MK33, Thermo electron corporation, China). Tissue culture plates (TCPS) without any film were used as a control.
3.1. Synthesis of allyl POSS–PMMA copolymers The allyl POSS–PMMA copolymer was synthesized via free radical polymerization using AIBN as the initiator, allyl POSS and MMA as monomers. The successful synthesis of copolymers was proved by 1H NMR. Fig. 1. is the typical 1H NMR spectra of 0.02 allyl POSS–PMMA copolymer recorded in CDCl3 with the relevant signals labeled. The characteristic resonance signals at 0.60 ppm, 0.91 ppm, 1.81 ppm, and 1.85 ppm were attributed to allyl POSS, while signals at 0.85 ppm, 1.03 ppm, 1.25 ppm, 1.60 ppm and 3.60 ppm belonged to PMMA. The Mn/Mw and polydispersity (PDI) of PMMA and allyl POSS– PMMA copolymers were calculated from GPC using THF as eluent. The characterization of PMMA and the copolymer is displayed in Table 1. GPC measurement indicated a unimodal molecular weight distribution for both the resultant copolymers and pure PMMA. The Mw/Mn for the 0.01 allyl POSS–PMMA copolymer was found to be 480,500/206,300 g/mol with a PDI of 2.38, typical for copolymers synthesized by free radical polymerization. The Mw/Mn for the 0.02 allyl POSS–PMMA copolymer was found to be 562,000/ 220,600 g/mol with a PDI of 2.49 (see Fig. 2.). For the pure PMMA, the Mw/Mn and PDI were found to be 448,600/210,400 g/mol and 2.13, respectively. Changes in chemical properties of PMMA to allyl POSS–PMMA copolymer due to introduce of POSS cage were examined using
4
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
Fig. 1. 1H-NMR spectrum of 0.02 allyl POSS–PMMA copolymer.
Table 1 Copolymerization of MMA and allyl POSS with free radical polymerization. Polymer Mna/g mol Mwb/g mol PDIc
1 1
PMMA
0.01 allyl POSS + PMMA
0.02 allyl POSS + PMMA
210,400 448,600 2.13
206,300 490,500 2.38
220,600 562,000 2.49
a The number-average molecular weight (Mn) were determined by gel permeation chromatography (GPC) performed in THF (1.0 ml/min). b Weight-average molecular meight (Mw) were determined by gel permeation chromatography (GPC) performed in THF (1.0 ml/min). c The molecular weight distributions were calculated as Mw/Mn.
FT-IR spectral analysis. The FT-IR spectrum of the allyl POSS– PMMA copolymer showed combined features for PMMA and allyl POSS (see Fig. 3). There were three strong vibration bands at 1637.0 cm 1, 1728.5 cm 1 and 1148.3 cm 1, attributable to C@O and CAOAC stretching vibrations from PMMA. The strong SiAOASi stretching vibration at 1115.2 cm 1 from the POSS cage indicated the existence of allyl POSS in copolymers [30], which demonstrated the successful synthesis of allyl POSS–PMMA copolymer. The X-ray scattering profiles of PMMA and allyl POSS–PMMA copolymer are shown in Fig. 4. The shape of the most intense peak reflects the ordered packing of polymer chains. The pure PMMA exhibited reflections 2h’s at 14° which is amorphous arrangement of polymer segment. For the 0.01 allyl POSS–PMMA copolymer, there were peaks 2h at 11° and 12° indicating regular arrangement of allyl POSS molecules. As the allyl POSS content increased to 0.02, the reflections (2h = ca. 11° and 12°) from allyl POSS component increased in intensity and sharpness while the new reflections at 8° and 9° were stronger and sharper. From this observation, it was confirmed that the allyl POSS cages formed the aggregation and crystalline domain.
Fig. 2. GPC chromatogram of 0.02 allyl POSS–PMMA copolymer.
3.2. Characterization of the material surfaces Fig. 5 shows the water contact angle (WCA) for the PMMA and allyl POSS–PMMA copolymer films with the extension of time from 5 s to 1440 s. In all cases, WCA was gradually decreased during the
Fig. 3. FT-IR spectra of PMMA and 0.02 allyl POSS–PMMA copolymer.
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
5
Fig. 4. X-ray diffraction patterns of PMMA and allyl POSS–PMMA copolymers. Fig. 7. Optical transmittance of the PMMA and allyl POSS–PMMA copolymer films.
Fig. 5. WCAs of the PMMA and allyl POSS–PMMA copolymer surfaces with the change of time. Fig. 8. TGA derivative plots of PMMA and allyl POSS–PMMA copolymers.
Fig. 9. DSC thermograms of PMMA and allyl POSS–PMMA copolymers.
Fig. 6. AFM images of films of (a) PMMA, (b) 0.01 allyl POSS–PMMA and (c) 0.02 allyl POSS–PMMA.
initial 900 s and reached plateau after that time. PMMA films showed hydrophobic property with WCAs of 94.7 ± 6.7° at 10 s and 74.5 ± 7.2° at 1440 s, while films consisting of allyl POSS had higher hydrophobicity with higher WCA values than PMMA film. The WCAs of allyl POSS–PMMA copolymer films (0.01 and 0.02)
6
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
Fig. 10. The cell viability assay of HLECs cultured on the surfaces of PMMA and allyl POSS–PMMA copolymers for 24 h. The absorbance of the diluted cell counting kit solution has been deducted from each data point and the statistical significance is indicated by different letters (p < 0.05).
were 101.8 ± 4.1° and 105.7 ± 3.0° at 10 s and 86.4 ± 3.8° and 88.2 ± 4.1° at 1440 s, respectively. The WCAs of the copolymers also showed that with the increase of allyl POSS from 0.01 to 0.02, the hydrophobicity slightly increased [25,31]. To further understand possible effect of the material composition on the surface morphology, we used AFM to study the morphology of spin-coated films. Photo images given in Fig. 6 showed the 3D images of the copolymers and pure PMMA films. It can be observed that the allyl POSS–PMMA copolymer films had a slightly higher roughness than pure PMMA film with RMS roughness of 2.96 ± 0.46 nm. As the adding of allyl POSS changing from 0.01 to 0.02, the RMS roughness of the films increased from 4.60 ± 0.52 nm to 5.84 ± 0.56 nm. The increase of roughness of the surface contributed to the hydrophobicity increase of copolymer films [32,33].
The transmittance of PMMA is also of great importance in its actual biomedical applications, especially used as intraocular lenses. So the transmittance of allyl POSS–PMMA copolymer films was also examined in this research, as shown in Fig. 7. It illustrated that the transmittance of pristine PMMA in visible region (400– 700 nm) was very excellent, with light transmission higher than 93%. However, the complex of allyl POSS in PMMA resulted in the decrease of the light transmission. The transmittances of 0.01 and 0.02 allyl POSS–PMMA copolymer films were respectively 91–93% and 90–92% in visible region. So the amount of doping allyl POSS should be controlled so as to keep transmittance of allyl POSS–PMMA copolymer films in a reasonable range. The thermal analysis was carried out by TGA and DSC. As seen in Fig. 8, the pure PMMA shows three-step thermal decomposition behaviors (160–260 °C, 260–310 °C and 310–400 °C), while the amphiphilic allyl POSS–PMMA hybrids exhibit clearly two-step thermal decomposition behaviors (160–260 °C and 260–400 °C), suggesting the altered thermal decomposition due to the incorporation of allyl POSS macromolecules [34]. The glass transition temperature (Tg) was taken as the inflection point of the heat flow of the second heating run. Fig. 9 showed the DSC heating traces of neat PMMA and allyl POSS–PMMA copolymer at 20 °C/min from the amorphous state. Neat PMMA exhibits a Tg around 115.7 °C. Incorporation of POSS comonomers into PMMA caused a slight decrease of Tg, while maintaining thermal stability. The Tg of 0.01 and 0.02 allyl POSS–PMMA copolymer were 111.5 °C and 102.3 °C, respectively. The slight decrease in Tg with an increase of allyl POSS content indicated that incorporation of allyl POSS macromolecule as a pendent group onto PMMA backbone facilitated the movement of polymer chains and resulted in crystallites with lower thermal stability [35]. 3.3. Cytotoxicity of the material surfaces The growth and proliferation of the HLECs on the surfaces of PMMA and allyl POSS–PMMA copolymer films were investigated
Fig. 11. Growth and morphology of HLECs stained with FDA after 24 h of incubation on (a) TCPS, (b) pristine PMMA film, (c) 0.01 allyl POSS–PMMA copolymer film, (d) 0.02 allyl POSS–PMMA copolymer film under fluorescence microscopy (the magnification is 10).
B. Wang et al. / Journal of Colloid and Interface Science 431 (2014) 1–7
to characterize the cell compatibility of the materials. Fig. 10 showed quantitative assessment of the cytotoxicity by CCK-8 assay over a period of 24 h. The results were the average of three replicate experiments. It was found that the cell viability of PMMA and allyl POSS–PMMA copolymer films was much lower than that of TCPS. It also could be seen that PMMA film had the lowest cell viability compared with TCPS and allyl POSS–PMMA copolymers. With the complex of allyl POSS, the cell viability increased from 0.409 ± 0.098 to 0.472 ± 0.103 and 0.498 ± 0.088 for 0.01 and 0.02 allyl POSS–PMMA copolymer films respectively. Surface properties have an important impact on not only cell viability, but also cell adhesion and proliferation [36,37]. The adhered HLECs on the surface were photographed with an inverted fluorescence microscope after FDA staining, and the results are shown in Fig. 11. After 24 h incubation, it could be seen that HLECs incubated on TCPS had changed their morphology to longer and bigger structures together with an obvious increase in number, suggesting the HLECs were in the growth status (Fig. 11(a)). The HLECs cultured on the PMMA and allyl POSS–PMMA copolymer films had undergone some degree of proliferation. However, the number of cells attachment was less comparing with TCPS. Simultaneously, the surfaces of allyl POSS–PMMA copolymer films had more HLECs and better spreading morphology than that on the surface of PMMA. This was consistent with the results of CCK-8. It has been reported that the surface chemistry of a material can mediate the cellular response to the material and affect cellular function on the surface including cell adhesion, proliferation and migration [38,39]. Surface wettability was the most important factor [40]. Studies of the influence of WCA on cell attachment and spreading further showed that cells exhibited poor attachment and spreading on a materials surface with a low WCA [41]. Cell membrane with hydrophobic property prefers hydrophobic material surfaces due to hydrophobic interactions [42,43]. The WCAs of all allyl POSS–PMMA copolymer films were higher than PMMA film, which promoted HLECs attachment and spreading. The adhesion of cells to different substrates surfaces is also affected by various chemical and physicochemical factors, such as roughness. According to sandwich theory of PCO [13–15], the promotion of HLECs adhesion on IOL can form a cell monolayer between IOL and posterior capsule, fill up the space and reduce the occurrence of PCO.
4. Conclusion Allyl POSS–PMMA copolymer was synthesized by radical polymerization and made into film as IOL material. FT-IR, XRD and 1H NMR measurements indicated the successful synthesis of allyl POSS–PMMA copolymer. By incorporating of ally POSS into the PMMA main chain results in good thermodynamic properties and high transparency of PMMA-based polymeric material. Characterizations of morphology and surface hydrophility suggested that the allyl POSS–PMMA copolymer film had a higher hydrophobicity and a higher roughness. The surface of allyl POSS–PMMA films had better cellular compatibility with HLECs and better spreading morphology than pure PMMA film. As a result, the presence of allyl POSS in PMMA in some extent promoted the epithelialization of HLECs on the surface and may reduce the occurrence of PCO, which may be of great interest being used as a novel IOL material.
7
Acknowledgments Financial supports from National Natural Science Foundation of China (81271703, 51203120), Natural Science Foundation of Zhejiang Province (LQ12E03001), Medical & Health Technology Program of Zhejiang Province (2013KYA133, 2014KYA149), the International Scientific and Technological Cooperation Projects (2012DFB30020) and the Specialized Research Fund for Science and Technology Major Projects of Higher Education of China (ZD2007006) are greatly acknowledged. References [1] E.J. Hollick, D.J. Spalton, P.G. Ursell, Arch. Ophthalmol. 117 (1999) 872. [2] J.W. Cheng, R.L. Wei, J.P. Cai, G.L. Xi, H. Zhu, Y. Li, X.Y. Ma, Am. J. Ophthalmol. 143 (2007) 428. [3] N. Li, X. Chen, J. Zhang, Y. Zhou, X. Yao, L. Du, M. Wei, Y. Liu, Ophthalmology 115 (2008) 830. [4] P.J. Morgan-Warren, J.A. Smith, Clin. Ophthalmol. 7 (2013) 1661. [5] H. Matsushima, H. Iwamoto, K. Mukai, Y. Katsuki, M. Nagata, T. Senoo, Expert Rev. Med. Dev. 5 (2008) 197. [6] D.M. Saylor, D.C. Richardson, B.J. Dair, S.K. Pollack, Acta Biomater. 6 (2010) 1090. [7] S. Sacu, R. Menapace, W. Buehl, G. Rainer, O. Findl, J. Cataract Refract. Surg. 30 (2004) 1875. [8] S. Argal, J. Clin. Ophthalmol. Res. 1 (2013) 113. [9] B. Amoozgar, D. Morarescu, H. Sheardown, Coll. Surf. B 111 (2013) 15. [10] S. Hazra, H. Palui, G.K. Vemuganti, Int. J. Ophthalmol. 5 (2012) 59. [11] S.M. Shah, D.J. Spalton, J. Cataract Refract. 21 (1995) 579. 579.J. [12] O. Findl, W. Buehl, R. Menapace, S. Sacu, M. Georgopoulos, G. Rainer, Ophthalmology 112 (2005) 2004. [13] R.J. Linnola, A. Holst, J. Cataract Refract. 24 (2001) 1509. [14] R.J. Linnola, J. Cataract Refract. 23 (1997) 1539. [15] S.M. Raj, A.R. Vasavada, S.R. Johar, V.A. Vasavada, Int. J. Biomed. Sci. 3 (2007) 237. [16] R.A. D’Sa, J. Raj, M.A.S. McMahon, D.A. McDowell, G.A. Burke, B.J. Meenan, J. Colloid Interface Sci. 375 (2012) 193. [17] M.J. Yang, Z.F. Di, J.K. Lee, J. Colloid Interface Sci. 368 (2012) 603. [18] S. Kang, M.J. Kim, S.H. Park, C.K. Joo, Euro. J. Ophthalmol. 18 (2008) 377. [19] X.D. Huang, K. Yao, Z. Zhang, Y.D. Zhang, Y. Wang, J. Cataract Refract. 36 (2010) 290. [20] L. Lin, Y. Wang, X.D. Huanga, Z.K. Xu, K. Yao, Appl. Surf. Sci. 256 (2010) 7354. [21] S.H. Phillips, T.S. Haddad, S.J. Tomczak, Interface Sci. 8 (2004) 21–29. [22] R.Y. Kannan, H.J. Salacinski, P.E. Butler, Acc. Chem. Res. 38 (2005) 879. [23] R.Y. Kannan, H.J. Salacinski, M.J. Edirisinghe, Biomaterials 27 (2006) 4618. [24] X.Y. Song, H.P. Geng, Q.F. Li, Polymer 47 (2006) 3049. [25] Y. Xue, H. Wang, D. Yu, L. Fen, Chem. Commun. 42 (2009) 6418. [26] H. Xu, B. Yang, J. Wang, S. Guang, C. Li, J. Poly. Sci. Part A: Poly. Chem. 45 (2007) 5308. [27] H. Liu, S. Zheng, Macromol. Rapid Commun. 26 (2005) 196. [28] P.T. Mather, H.G. Jeon, A. Romo-Uribe, T.S. Haddad, J.D. Lichtenhan, Macromolecules 32 (1999) 1194. [29] B. Seurer, E.B. Coughlin, Polym. Prepr. 46 (2005) 738. [30] Y.L. Liu, M.C. Tseng, M.H. Fangchiang, J. Poly. Sci. Part A: Poly. Chem. 46 (2008) 5157. [31] A. Tuteja, W. Choi, M. Ma, J.M. Mabry, S.A. Mazzella, G.C. Rutledge, G.H. McKinley, R.E. Cohen, Science 318 (2007) 1618. [32] Y. Tian, L. Jiang, Nat. Mater. 12 (2013) 291. [33] K.J. Kubiaka, M.C.T. Wilsona, T.G. Mathiab, P. Carvalc, Wear 271 (2011) 523. [34] C.K. Kim, B.S. Kim, F.A. Sheikh, U.S. Lee, M.S. Khil, H.Y. Kim, Macromolecules 40 (2007) 4823. [35] Y. Yani, M.H. Lamm, Polymer 50 (2009) 1324. [36] S.S. Li, D.Y. Yang, H.Y. Tu, H.T. Deng, D. Du, A.D. Zhang, J. Colloid Interface Sci. 402 (2013) 284. [37] B. Cortese, M.O. Riehle, S. D’Amon, G. Gigli, J. Colloid Interface Sci. 394 (2013) 582. [38] Z. Zheng, F.F. Bei, H.L. Tian, G.Q. Chen, Biomaterials 26 (2005) 3537. [39] Z.W. Dai, X.H. Zou, G.Q. Chen, Biomaterials 30 (2009) 3075. [40] N.J. Hallab, K.J. Bundy, K. O’Connor, R.L. Moses, J.J. Jacobs, Tissue Eng. 7 (2001) 55. [41] L.H. Zhang, D.W.Y.S. Chen, X.L. Wang, G.W. Zhao, H.Y. Wang, C.Z. Huang, Appl. Surf. Sci. 255 (2009) 6840. [42] J.D. Mendelsohn, S.Y. Yang, J.A. Hiller, A.I. Hochbaum, M.F. Rubner, Biomacromolecules 4 (2003) 96–106. [43] B.L. Wang, J.L. Wang, D.D. Li, K.F. Ren, J. Ji, Appl. Surf. Sci. 258 (2012) 7801.