The Analysis of Surface Treatment of PDMS on Prostate Cancer and Smooth Muscle Cells

Transcription

1 SPIE Smart Materials, Nano- and Micro-Smart Systems Symposium, Melbourne, Australia, Dec The Analysis of Surface Treatment of PDMS on Prostate Cancer and Smooth Muscle Cells Cory Huggins 1, Smitha M. N. Rao 2, Kytai Nguyen 1, and J.-C. Chiao 1,2 1 Deptartment of Bioengineering, 2 Department of Electrical Engineering The University of Texas at Arlington 416 Yates St., Arlington, TX 76019, USA ABSTRACT The analysis of cellular activity when exposed to polydimethylsiloxane (PDMS) is necessary as this material has been used in various applications such as tissue engineering and microfluidic devices for cellular studies due to the polymer s unique mechanical properties. In this particular study, we investigated the effects of corona surface treated PDMS with different cross-linker ratios on cellular activities by analyzing prostate cancer cell (PC-3) and vascular smooth muscle cell (VSMC) adhesion and proliferation. Both cell lines were subjected to a thin PDMS layer immediately after and 24 hours after corona treatment. The results indicated steady cell adhesion and proliferation rates for both smooth muscle and prostate cancer cells when seeded onto PDMS 24 hours after corona surface treatment, but significantly less cell adhesion when seeded immediately after activation and controls (PDMS without any treatment). These results would allow future PC-3 and VSMC experiments to be performed in a PDMS environment that is not detrimental for adhesion and proliferation. Keywords: Polydimethylsiloxane, corona treatment, cell adhesion, cell proliferation 1. INTRODUCTION Polydimetylsiloxane (PDMS) is an attractive polymer used throughout many industrial and medical applications today due to its good electrical/mechanical properties and low surface energy. This is due to its ability to undergo chain rearrangement after the alteration of surface chemistry in order to restore its hydrophobic nature [1,2]. This polymer has also proved to be stable when exposed to a wide range of temperatures [3]. Although PDMS consists of many attractive properties, its use in cell studies has been limited. PDMS has been modified in order to enhance cell adhesion and proliferation for cellular studies. Modification of PDMS using oxygen plasma, which involves rending the polymer hydrophilic, has become a common practice [4]. Corona surface treatment has also been applied as an effective method of increasing oxygen concentration on the surface of PDMS [8,9]. The use of corona discharge aids in the production of heat and UV radiation as well as by-products such as ozone [10]. This discharge results in an oxidized PDMS surface which contains siloxane groups and has the ability to restore its original nature by chain re-orientation [11, 12]. It has also been concluded that the extent of corona exposure to PDMS will directly affect its recovery rate [3]. The corona treatment was used because it is inexpensive, easy to conduct, and provides the necessary alterations to a hydrophobic polymer. PDMS has been used quite frequently throughout many cell studies in reference to how the alteration in its mechanical properties affects cell adhesion [5-7]. In this paper, we present the results of the effects by corona treatment on PC-3 and vascular smooth muscle cells. The contact angle measurements of PDMS were obtained immediately after activation, 24 hours after activation and without activation. This data would aid in the analysis of its hydrophobic/hydrophilic nature and provide additional information of future cell activity. Thin layers of PDMS with different cross-linker ratios of 10:1, 30:1 and 50:1within well plates were exposed to corona surface treatment. Each cell type was seeded at the respective cross-linker ratios immediately after activation, 24 hours after activation and on untreated PDMS termed throughout the remainder of this paper

2 Activated Immediately, Activated and Non-Activated, respectively. Cell adhesion and proliferation over a given time period was analyzed using PicoGreen DNA assays to assess the total cell DNA that is correlated to the cell number in the sample. Microscopy analysis was also performed in order to visualize the effects on cell adhesion and proliferation over a given time period. These results would aid in future microscale device fabrication that has the potential to be used for a variety of cell studies. 2.1 Substrate Fabrication 2. MATERIALS AND METHODS PDMS is composed of a base and a curing agent that is typically mixed 10:1 (by weight), respectively. In this experiment, PDMS was mixed with cross-linker ratios of 10:1, 30:1, and 50:1. A thin layer of the PDMS mixture was placed at the base of 24 well plates. The PDMS was allowed to cure for 48 hours at room temperature, which also aided in the degassing process. Then, the respective well plates were activated with corona surface treatment and immediately seeded with cells as the Activated Immediately group. Other wells were activated via corona treatment and were placed aside for 24 hours prior to cell seeding as the Activated group. A group of well plates marked as Non-Activated was the PDMS without any further treatment. The corona treatment method is simple, low cost and time efficient compared to the layer-by-layer self-assembly (LBL) method. The well plates were sterilized under ultraviolet (UV) light exposure for 30 minutes which in fact caused chain scission, but the original nature is virtually restored instantly [13]. 2.2 Surface Analysis Figure 1 displays a sessile drop goniometer, manufactured by Rame-Hart Inc., which was used to characterize the surface of each cross-linker ratio of PDMS before and after activation. Contact angle measurements for the Non-Activated PDMS were also calculated using this method. In all three instances, three measurements were taken per sample and the average values and deviations were recorded. Lens Reservoir Stage Light Source Figure 1: A photo of the contact angle measurement setup. A sessile drop goniometer was used in order to obtain the contact angles for each cross-linker ratio after their respective surface alterations.

3 2.3 Cell Culture PC-3 and VSMC were cultured in complete Dulbecco s-modified Eagle s medium (DMEM) which contained 10% bovine serum and 1% penicillin-streptomycin. Each cell line was grown to 80% confluency before the cells were seeded on the well plates. 2.4 Cell Attachment and Proliferation The PC-3 and VSMC were seeded in well plates as mentioned above at the seeding density of and , respectively. Cells were then incubated at 37ºC and allowed to grow over various time points. At a specific time (1, 2, 3 and 4 days after seeding), cells were lysed with 1% Triton for 40 minutes, and the cell lysis samples were analyzed for the cell growth using PicoGreen DNA assays (Invitrogen) following the manufacturer s instructions. 3. EXPERIMENTAL RESULTS Contact angle measurements were obtained for each of the analyzed surfaces as shown in Table 1. The result from three samples for each test configuration indicates that the use of the corona treatment device renders the PDMS surface layer hydrophilic, immediately. The distribution of water molecules throughout the surface contained a contact angle of 0 for cross-linker ratios of 10:1, 30:1 and 50:1. Activated PDMS had similar contact angle measurements, 68 and 61, for cross-linker ratios of 10:1 and 30:1, respectively. A cross-linker ratio of 50:1 proved to be the most hydrophobic of the Activated PDMS with a contact angle of 90. The Non-Activated surface showed a steady increase in contact angle with respect to the cross-linker ratio. Crosslinker ratios of 10:1, 30:1 and 50:1 obtained contact angles of 115, 124 and 129, respectively. With this knowledge, we are able to analyze the relationship between the hydrophobic/hydrophilic polymer with respect to cell adhesion and proliferation. Table 1: Contact angle measurements for the variations in cross-linker ratios of PDMS. 10:1 30:1 50:1 Activated Immediately 0 ±3 0 ±3 0 ±3 Activated 68 ±3 61 ±3 90 ±3 Non-Activated 115 ±3 124 ±3 129 ±3 The PC-3 cells proved to grow on various conditions of PDMS over a 3-day period. Figure 2(a) shows PC-3 cell growth at Day 3 on the Activated surface with a cross-linker ratio of 10:1. The cells are attached to the PDMS surface and dividing at a rate which displays affinity towards the activated surface. For the Activated Immediately surface, Fig. 2(b) shows some cell attachment, but includes a few clusters of cells that are detached. These are most likely a combination of cells undergoing mitotic division as well as deceased cells. Further analysis of this particular surface verifies a decrease in cell proliferation as time increases when compared to the Activated surface. Fig. 2(c) shows large clusters of cells on the Non-Activated PDMS. There is much less uniformly cell attachment when compared to the previous two surfaces. The image in Fig. 2(d) is the positive control group in the base of the well plate without PDMS, which shows similarities to the image in Fig. 2(a). These results demonstrate that cells grown on the Activated PDMS are similar to those grown on the tissue culture well plates, suggesting Activated PDMS is better for cell growth compared to other groups.

4 (a) (b) (c) (d) Figure 2: Effects of PC-3 cell adhesion and proliferation on a 10:1 cross-linker ratio of PDMS at Day 3. (a) Activated, (b) Activated Immediately, (c) Non-Activated, and (d) Tissue-cultured treated well plates as positive controls. Cell proliferation for each analyzed surface at their respective cross-linker ratios proved to be insufficient at Day 1 as shown in Fig. 3. The data shows poor attachment and growth which ranged from 14.4% 17.4% of the control group. Also, there is not a distinct variation between the treatments that were administered as well as the cross-linker ratios. The adhesion and proliferation of PC-3 cells of the Activated Immediately and Non- Activated groups on Day 2, Fig. 3(b), decreased when compared to Day 1. Each cross-linker ratio on Day 2 for the Activated group obtained an increase in concentration, even through the percent of control for 50:1 shows a decrease from the previous day. This was due to the rapid proliferation of the control group. Shown in Fig. 3(c), a cross-linker ratio of 10:1 with Activated conditions proved to have very similar results obtained by the control group on Day 3. Cross-linker ratios of 30:1 and 50:1 in this category proved to be effective displaying cell growth of approximately 90% of the control group. This signifies the importance of Activated PDMS on cellular activity over a 3-day period. The Activated Immediately group did not display a direct correlation between the cross-linker ratios while obtaining values of 55%, 59% and 46% for cross-linker ratios of 10:1, 30:1 and 50:1, respectively. One important factor is that there is not a great disparity of values regarding cross-linker ratios for both the Activated and Activated Immediately groups. The Non-Activated PDMS showed a significantly smaller amount of cell growth after three days of incubation compared to the controls (tissue culture plates). The amount of cell growth from the PDMS decreased with time. The cellular proliferation on Non-Activated PDMS was not dependent on the cross-linker ratio as well. Due to the

5 tremendous amount of detached clusters (Fig. 2) and little cell growth (Fig. 3), Non-Activated PDMS will not be an applicable substrate for our future cell studies. (a) (b) (c) Figure 3: PC-3 cell counts obtained at (a) Day 1, (b) Day 2 and (c) Day 3 for each analyzed surface at their respective cross-linker ratios. In the analysis of the smooth muscle cells at Day 1, Fig. 4 (a) displays similar results that were obtained in the PC-3 cell study pertaining to the percent of control. The percent of control in reference to attachment and proliferation at each cross-linker ratio ranged from 10.4% 16.7% for Day 1. This was expected due to the minimal time frame allowed for cellular adhesion and subsequently proliferation. Day 2 showed a drastic

6 increase, up to 51.3% of the control, in smooth muscle cell attachment for the Activated PDMS at a crosslinker ratio of 30:1. A cross-linker ratio of 10:1 also proved advantageous to cell adhesion while obtaining results very similar to 30:1. The overall Activated Immediately group on Day 2 proved inferior to the Activated group showing a decrease with respect to cross-linker ratios of 10:1 and 30:1, with values of 35% and 43.2%, respectively. Also, a cross-linker ratio of 50:1 achieved a slight increase with a value of 36.3% of the control group. Day 3 displayed another increase in VSMC attachment and proliferation for the Activated group, but it was a cross-linker ratio of 30:1 that obtained the highest percent of control at 78.5%. Unlike the previous day, there was not a significant difference between cross-linker ratios of 10:1 and 50:1. There was not a significant difference between the cross-linker ratios of the Activated Immediately group which contained values of approximately 38%. Day 4 showed an even distribution for the Activated group between cross-linker ratios of 10:1 and 30:1 which included approximately 80% attachment value as compared to the control group. Again, the Activated Immediately PDMS proved to be substantially less sufficient for VSMC attachment and proliferation as compared to Activated PDMS. One defining characteristic of this particular study was that the Non-Activated PDMS surface proved to be an insufficient environment for VSMC due to the extremely minimal cell attachment and proliferation rate over a four day period. (a) (b) (c) (d) Figure 4: Vascular Smooth Muscle cell counts obtained for each analyzed surface at their respective crosslinker ratios where Days 1, 2, 3 and 4 are represented by (a), (b), (c) and (d), respectively.

7 4. DISCUSSION Activating the surface of the PDMS proved to be beneficial for both prostate cancer cell PC-3 and vascular smooth muscle cell adhesion and proliferation over a longer time. This was somewhat predicted when the use of the corona surface treatment proved to alter the hydrophobic nature of the polymer. One concern after this treatment was how the nature of the polymer would react with proteins within the complete media as well as the cellular adhesion molecules. This leads us to believe there is an extensive delay of protein adsorption on the corona treated PDMS. In the PC-3 analysis, there was approximately 20% attachment when compared to the control in the Activated group. We believe the decrease in percent is due to adhesion of proteins within the media. As you can see, by Day 3 the PC-3 cells that adhered onto the surface of Activated PDMS are proliferating at a rate comparable to that of the control group. In reference to the Activated Immediately and Non-Activated conditions, we suspect that the Activated Immediately surface contains a surface layer that too hydrophilic for proper protein and cellular adhesion while the Non-Activated surface is too hydrophobic for these proteins or the cells to adhere and grow normally. Figure 3 displays an elevated hydrophobic surface is much more detrimental to cell attachment than a prominent hydrophilic surface. Overall, the Activated surface by corona treatment proves to be advantageous for prostate cancer cell adhesion and proliferation over a three-day period. The VSMC results over a four day study concluded that the 10:1 and 30:1 cross-linker ratios of PDMS proved to be the best method for analyzing the cell adhesion and proliferation rate. A large distinction from the crosslinker ratio of 50:1 was obtained in the Activated analysis, which infers that it is not an ideal cross-linker ratio for cell attachment in reference to the other cross-linker ratios. Also, the Activated Immediately surface is adequate for cell attachment and proliferation, but is clearly not the best method for obtaining superlative results. The Non-Activated surface once again proved to have the least cell number of the three analyzed surface. The minimal cell growth for each cross-linker ratio might be due to the hydrophobic nature of Non- Activated PDMS. Similar to our observation, Brown et. al. also found that untreated PDMS did not support VSMC adhesion [10]. From these results, we can infer that there is not a direct correlation between the cross-linker ratios on PC-3 adhesion and proliferation rates over a three day period. We can also infer that VSMC adhesion and proliferation is dependent upon cross-linker ratios when subjected to the Activated PDMS. Finally, different cell types will react differently to PDMS modified with various cross-linker ratios. 5. CONCLUSIONS The use of corona treatment increases the effect of PC-3 and VSMC cell attachment and proliferation on PDMS. Also, the elapsed time period before cell seeding on activated PDMS using corona treatment has also proved to be extremely important on cellular adhesion and proliferation. The cross-linker ratio of PDMS does not have a great effect on PC-3 adhesion over a three day period. However, it does have an effect on VSMC, especially for those grown on Activated PDMS. Further analysis of protein adsorption will be investigated in order to obtain a more thorough explanation of the cellular activity at the PDMS surface. These results will potentially aid further development of microfluidic devices and analysis of PC-3 and VSMC when exposed to PDMS surfaces. ACKNOWLEDGEMENTS This work is supported by AFOSR FA , Texas MrCeDm, and NSFREU ECS

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