Design of Smart Surface by Nanometric Grafting of NIPAAm with Benzophenone Initiator under UV Radiation

Introduction

Poly-N-Isopropylacrylamide and its copolymers in due to its high-speed phase transition from liquid to solid and being critical dissolution temperature of 32 °C can be used in the field of medical science as well as drug delivery system and tissue engineering. At high temperatures of 32 °C, the material shows a solid and hydrophobic state; and at temperatures below 32 °C, the polymer shows fully hydrated and hydrophilic properties. PNIPAAm was synthesized in 1957 by Wooten.¹ With this smart polymer, surface modification of materials can be used in cell sheet engineering. ^{2, 5} Different methods for surface modification of polymers like polyethylene, polypropylene, polystyrene and polyethylene terephthalate with PNIPAAm grafting with chemical, physical methods such as gamma-ray exposure, plasma, ozone and ultraviolet and electron beam is used, each has advantages and disadvantages. ^6,9 In all such methods, a radical is created on the surface; and during the collision with monomer, the polymerization process occurs.

Ultraviolet irradiation method in terms of simplicity and low cost as good as possibility of developing is a suitable method for biomaterial surface modification. ^10,11  Factors such as the distance of radiation, absorption intensity, the wavelength used appropriately to the initiator, thickness and usual factors such as degassing, substrate and initiators and sensitizers contributed in the ultraviolet radiation.¹²  Of course, this method is required for optical sensitizers or initiators such as anthraquinone¹³ or benzophenone. ¹⁴ Selected solvents used in spectroscopy and polymerization with ultraviolet radiation are very import. In this study, benzophenone initiator was used for grafting PNIPAAm on the polystyrene surface by UV radiation. Finally, a nanometeric uniform thickness was obtained. Also, the nanometeric thickness of grafting was shown to have an important effect in the process of adhesion and separation of cells and cell sheets. In this study, technical knowledge was also provided to achieve intelligent surfaces with nanometric thickness by using this type of radiation. 

Experiment

Materials

Polystyrene dishes (orange co) with the dimension of 1×1 cm² and 1 mm thickness, ethanol and methanol (Merck co), NIPAAm (Aldrich co), n-hexane (Merck co), distilled water and tissue culture polystyrene (TCPS), and epithelial cells (pastor institute, iran) were used in this study. Polystyrene dishes were put in the solution of ethanol-methanol with a 50/50 ratio for 24 hours to dissolve impurities and the oils existing on the surface of the dishes. After bringing out the dishes from the solution, they were washed by distilled water. For recrystallization of NIPAAm, 10.3 grams of NIPAAm (Aldrich Co) were dissolved in 125 ml n-hexane and the solution was inserted into a refrigerator to make the NIPAAm ready for grafting.

Sample preparation

The NIPAAm monomers were dissolved in water solvent (30%W) with to constant ratio of benzophenone initiator (3% w/v) (Fluka co). The samples were degassed by nitrogen (2bar mass flow rate) for 30 minutes. This process was followed to increase the efficiency of the free radical polymerization (deoxygenating). Then, the solutions were poured in a plastic washer (diameter=1cm and height=3 mm) that was attached to the polystyrene substrate. The samples with solution were exposed by a UV radiation source (black light, 160 W, 365 nm: Philips) for 30 min. Irradiations were carried out in the air under ambient conditions; then, the samples were brought out and washed by distilled water and were put in distilled water for 72 hours and Soxhleted for removing the ungrafted monomer, then were taken out for analysis.

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR)

The samples were examined by ATR-FTIR (Themo niocolet, nexus 870 ft-sr-USA) before and after adjustment, and there were put under instrument to investigate. 

Surface Morphology Study

The surface characteristics of modified and unmodified films were studied with the help of SEM (Cambridge Stereo scan, model S-360) to analyze the changes in the surface morphology. The films were first coated with a gold layer (Joel fine coat, ion sputter for 2 hours) to provide surface conduction before their scanning. The surface topology characteristics and the thickness of modified as well as the unmodified films were studied with the help of AFM (TMX 2010 and the nano-scurf easy scan 2 contact model) to analyze the changes in the surface topology. 

Contact Angle Analysis

Surface’s static contact angle of the samples was investigated by the contact angle measuring device (Kruss G10) following the sessile drop method. The formed contact angle would be the angle between the solid/liquid and the liquid/steam joint surface. In order to review the sample surface’s hydrophilic/hydrophobic behavior in high and low temperatures, a better sample was considered in two different temperatures of 4°C and 37°C and the contact angles were measured under these temperatures.

Biocompatibility Study

Aliquots of cell suspension in the RPMI medium including 300,000 SW742 epithelial cells were seeded on a 6-multiwell cell culture plate (orange), which was pre-coated with samples. The plate was incubated in a incubator (37 °C, CO2) in 3 h for cell attachment, followed by rinsing off the loosely attached cells with the phosphate buffer solution, and by adding 2 ml of fresh medium because of the cell culture in an incubator (37 °C, CO2) for 7 days. The proliferation of cells was determined for measuring the viable cell number by MTT assay. The MTT tetrazolium compound was reduced by living cells into a colored formazan product that was soluble in a tissue culture medium. The quantity of formazan product was directly proportional to the number of viable cells in the culture. The assays were performed by adding 1 ml of MTT solution (Sigma) and 9 ml fresh medium to each well after aspirating the spent medium, and incubating at 37°C for 4 h with protection from light. The colorimetric measurement of formazan dyeing was performed at a wavelength of 570 nm using a microplate reader (RAYTO).

For cell detachment, SW742 cells were seeded onto the samples at the density of 1,000,000 cells, and were cultured at 37°C under a humidified atmosphere of 5% CO₂. The cell detachment was evaluated by incubating the cultures at 4°C for up to 60 min. The culture medium that included the detached cells was transferred to a new well. The number of detached cells and the attached cells to the original well was determined by the MTT assay.

Results and discussion

ATR-FTIR Analysis

ATR-FTIR spectra results of the unmodified and the UV radiation modified polystyrene samples have been shown in Figure 1. The NIPAAm grafted by the UV radiated polystyrene ATR-FTIR spectrums have been shown below Figure 1B. The PNIPAAm picks characteristic includes 1601 cm^-1 which indicates –NH groups and 1730-1830 cm^-1 which indicates C=O groups and 3025 cm^-1 which indicates CH₃ groups and 3443 cm^-1 which indicates NH groups in PNIPAAm. All these picks are found in PNIPAAm grafted polystyrene samples. This conclusion shows grafting between the PNIPAAm with the polystyrene surface through UV radiation coating activation .

 

Table 1: Click here to View table

 

Surface Morphology Study

The microscopic images for investigating the modified samples through UV radiation have been shown in figures 2 and 3. These images showed the PNIPAAm grafting on the polystyrene surface. Figure 2A is the obtained AFM image from the unmodified polystyrene samples. The surfaces topography and the created graft thickness on surface are shown in the AFM images. Figures 2B-D show the surface topography for the grafted sample in the different scales. The graft thickness average for the sample grafting was about 300-600 nm .

Table 1: Click here to View table

 

Figure 3 is the obtained SEM image from the unmodified and modified polystyrene samples. Figure 3A is the obtained SEM image from the unmodified polystyrene sample (1000 ×). Figures 3A and 3B are the obtained SEM images from the modified polystyrene samples at different magnification. The grafted surfaces with the PNIPAAm  are shown in the SEM images.

Table 1: Click here to View table

 

Contact Angle Analysis

In the contact angle measurement process, the measured angle of normal polystyrene adjusted by UV radiated PNIPAAm surface samples in 4 °C and 37 °C temperatures has been shown in Table 1. The tabular data of the best grafted sample (with 40% of NIPAAm concentration resolved in  the solvent of 9:1 (v/v) water/methanol ) indicate the fact  that in 4°C and 37°C,  the samples show different contact angles which is also another reason for the existence of PNIPAAm grafting onto polystyrene surfaces.

Table 1: Contact angle for the unmodified and modified samples

	Normal	Grafted samples
T(°C )	θH2O	θH2O
25	90 ± 3.2o	60 ± 0.4o
37	90 ± 3.2o	63.2 ± 0.3o
4	90 ± 3.2o	48.5 ± 1.9o

The contact angles average 48.5° and 63.2° have been calculated for 4°C and 37°C temperatures. The result indicated a contact angle decrease below the temperature of 32°C (4°C), and it showed the hydrophilic surface feature. The contact angle increased in above 32°C temperature (37°C) which also showed the hydrophobic surface feature.

Biocompatibility Results

The biocompatibility data demonstrated that the grafted samples under UV radiation supported epithelial cell adhesion and proliferation; and the cells also maintained suitable viability (Table 2). After cultured for 7 days on grafted samples, many cells were alive, suggesting that the grafted samples were suitable for cell attachment and proliferation; and the viability was about 80%. When the cells were placed outside the incubator and the medium was cooled from 37°C to 4 °C, almost all of them were alive (the viability was about 70%) .

Table 2: MTT analysis of the samples

Figure 4A shows a good cell growth on the grafted samples surface at the physiological temperature of 37°C. Figure 8b shows cells growth detached from the grafted samples surface spontaneously, in the absence of enzymes (trypsin/EDTA). Cell detachment efficiency from the grafted samples was high. In contrast, cells growth on theTCPS dishes did not show such temperature-dependent cell sheet detachment. After a longer period of cell cultivation for 7 days, confluent cells formed a continuous monolayer cell sheet on the surface of the grafted samples. The cell sheet was spontaneously detached from the surface of the thermo-reversible grafted samples when cooled to 4°C without treating by any enzymes. As shown (Figure 4B), the detachment of the cell monolayer created a cell monolayer. After 60 min incubation at 4°C, a monolayer cell sheet could be lifted up from the edge upon mild perturbation of the medium. A living cell sheet, detached from the culture surface, could be obtained within 60 min. Such results demonstrated that cold treatment effectively released the cell sheet from the plate without considerable damage of the cell–cell connections.

Table 1: Click here to View table

Conclusion 

The polymer grafting on a polystyrene surface with chemical initiator under UV radiation was studied in this article. The ATR-FTIR spectrum showed the existence of the grafted polymer on polystyrene surfaces. The SEM images showed the grafted surfaces morphology in different magnifications; we could clearly observe and compare our graft increases. The topology of the surfaces shown in the AFM images also approved the claim. The graft thickness of the grafted samples in this study was about 300-600 nm. The contact angles 48° and 63° obtained in 4°C and 37°C temperatures. Thermo-responsive polymers were grafted to dishes covalently, which allowed the epithelial cells to attach and proliferate in 37°C. Also cells (cell sheet) were detached spontaneously when temperature decreased below 32_°C, without using enzymes. Also MTT analysis showed good viability of grafted samples. This characteristic proved that such type of grafted materials had potential as biomaterials for cell sheet engineering.

References

1. Heskins M, Guillet J.E. Solution properties of poly(N-isopropylacrylamide). J. Macromol. Sci. Chem. 1968; A28, 1441–1455.
2. Ricardo M.P. da Silva, Joa˜o F. Manoand Rui L. Reis. Smart thermoresponsive coatings and surfaces for tissue engineering : switching cell-material boundaries. Trends in Biotechnology . 2007;25(12):577-583.
3. Yakushiji T, Sakai K, Kikuchi A, Aoyagi T, Sakurai Y, Okano T. Graft  architectural effects  on  thermo-responsive wettability changes of poly(N-isopropylacrylamide)-modified Surfaces. Langmuir. 1998; 14 (16):4657–4662.
4. Kikuchi A, Okano T. Nanostructured designs of biomedical materials applications of cell sheet engineering to functional regenerative tissues and organs. J Control Release. 2005;101(1–3):69–84.
5. Galaev I.Y, Mattiasson B. Stimulus Responsive Surfaces: Possible Implication for Biochromatography, Cell Detachment and Drug Delivery, Landes Bioscience . 2003; chapter 6:116-129.
6. Biazar E ,et al. Cell engineering: nanometric grafting of  poly-N-isopropylacrylamide onto polystyrene flm by different doses of gamma radiation. International Journal of Nanomedicine. 2010;5: 549–556.
7. Curti P.S, De Moura M.R, Radovanovic E, Rubira A.F, Muniz E.C. Surface modification of polystyrene and poly(ethylene terephtalate) by grafting poly(N-isopropylacrylamide). Journal of materials science: materials in medicine. 2002;13:1175-1180.
8. Joeng B, Gutowska A. Lessons from nature: stimuli-responsive polymers and their applications. Trends Biotechnol. 2002;20:305–310.
9. Akiyama Y, Kikuchi A, Yamato M, Okano T. Ultrathin poly(N-isopropylacrylamide) grafted layer on poly(styrene) surfaces for cell adhesion/detachment control. Langmuir. 2004;20(13):5506–5511.
10. Delivopoulos E , Murray AF, Curtis JC. Effects of parylene-C photooxidation on serum-assisted glial and neuronal patterning. Journal of Biomedical Materials Research Part A. 2010; 94A(1):47–58.
11. Delivopoulos E, Murray AF, MacLeod NK, Curtis JC. Guided growth of neurons and glia using microfabricated patterns of parylene-C on a SiO2 background. Biomaterials. 2009 ;30 (11):2048-58.
12. Jianping Deng, Lifu Wang, lianying Liu,Wantai Yang. Developments and new applications of UV-induced surface graft polymerizations. Progress in Polymer Science .2009; 34(2):156-193.
13. Geuskens G, Etoc A, Di Michele P. Surface modification of polymers VII.: Photochemical grafting of acrylamide and N-isopropylacrylamide onto polyethylene initiated by anthraquinone-2-sulfonate adsorbed at the surface of the polymer.  European Polymer Journal. 2000;36(2):265-271.
14. Bousquet J.A, Haidar B, Fouassier J.P, Vidal A. Photocrosslinking of elastomer systems—III Ultraviolet light induced reactions in EPDM and EPR blended with or modified by grafting of derivatives of benzophenone.  European Polymer Journal.1983;19(2):135-142.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.