ARTICLE pubs.acs.org/Langmuir
Stimulus-Responsiveness and Drug Release from Porous Silicon Films ATRP-Grafted with Poly(N-isopropylacrylamide) Roshan B. Vasani,† Steven J. P. McInnes,† Martin A. Cole,‡ Abdul Mutalib Md Jani,† Amanda V. Ellis,† and Nicolas H. Voelcker*,† † ‡
School of Chemical and Physical Sciences, Flinders University, Bedford Park, SA 5042, Australia The Interdisciplinary Nanoscience Centre (iNANO), Aarhus University, Aarhus C 8000, Denmark
bS Supporting Information ABSTRACT: In this report, we employ surface-initiated atom transfer radical polymerization (SI-ATRP) to graft a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM), of controlled thickness from porous silicon (pSi) films to produce a stimulus-responsive inorganicorganic composite material. The optical properties of this material are studied using interferometric reflectance spectroscopy (IRS) above and below the lower critical solution temperature (LCST) of the PNIPAM graft polymer with regard to variation of pore sizes and thickness of the pSi layer (using discrete samples and pSi gradients) and also the thickness of the PNIPAM coatings. Our investigations of the composite’s thermal switching properties show that pore size, pSi layer thickness, and PNIPAM coating thickness critically influence the material’s thermoresponsiveness. This composite material has considerable potential for a range of applications including temperature sensors and feedback controlled drug release. Indeed, we demonstrate that modulation of the temperature around the LCST significantly alters the rate of release of the fluorescent anticancer drug camptothecin from the pSi-PNIPAM composite films.
’ INTRODUCTION Porous silicon (pSi) is a nanostructured material with a surface area of up to 800 m2 g1 that is commonly produced from bulk single crystal silicon by electrochemical anodization in hydrofluoric acid.1,2 An attractive property of pSi is that the size and type of pore generated can be altered by controlling certain factors such as the doping of the silicon wafers (p-type or n-type), the resistivity, the current density applied and the type of electrolyte used. By tuning these parameters, macroporous (pore size >50 nm), mesoporous (pore size 550 nm), and microporous (pore size LCST, 40 °C). This distinct change in the contact angle of about 23° is comparable to the value found in literature, where for similar PNIPAM thicknesses on flat pSi surfaces Li et al.62 found the change in contact angle around the LCST to be in the range of 2530°. Additionally, Cunliffe et al.63 also reported changes of contact angle in the same range for PNIPAM grafted from the surface of a glass slide. These results demonstrate that the temperature induced wettability change in PNIPAM persists when grafted from porous films. The forces between a clean silicon nitride AFM tip and the PNIPAM-grafted pSi surface were measured on N1 type pSiPNIPAM in Milli-Q water. Measurements were taken at both 25 and 50 °C. Figure 4a shows the force versus distance curves (normalized for comparison) obtained when the tip was retracted from the surface of the pSi-PNIPAM sample. At temperatures above the LCST (50 °C), a small adhesion force (approximately 0.22 ( 0.05 nN) between the AFM tip and the surface was observed. This adhesion is most likely caused by hydrophobic effects between the tip and the polymer above the LCST in aqueous medium. In contrast, adhesion forces between the tip and surface were absent when the tip was retracted from the surface at temperatures below the LCST of PNIPAM (20 °C).
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Figure 4. (a) Force versus distance retract curves measured in water for pSi-PNIPAM at 50 °C (9) and 20 °C ([). Typical ΔEOT% versus time measurements obtained above and below the LCST of PNIPAM, using (b) P1-type BiBAPTES functionalized pSi (control), (c) P1-type pSiPNIPAM, and (d) N1-type pSi-PNIPAM. The arrows indicate the time at which the pump was switched from hot to cold water or vice versa.
The approach curve at this temperature (Supporting Information Figure S4) did however show steric compressive interactions of the tip with a well-hydrated PNIPAM coating.64 The force spectroscopy results therefore corroborate the contact angle experiments, confirming the phase transition of the grafted polymer. The refractive index of PNIPAM has been shown to reversibly increase when heated above and decrease when cooled below the LCST.47 This modulation in refractive index allows the study of the volume-phase transitions of the polymer within the pSi matrix using IRS as shown by Segal et al.15 The pSi-PNIPAM samples were clamped into a flow cell, and the volume-phase transition was induced by flowing Milli-Q water of different temperatures (25 and 40 °C, respectively) over the sample. A pSi sample functionalized with BiBAPTES was used as a control to investigate the effect of temperature change on the refractive index of the porous layer itself. Figure 4 shows typical percentage change in EOT (ΔEOT%) sensorgrams of a control sample without PNIPAM (Figure 4b), a P1-type pSi-PNIPAM sample (Figure 4c), and an N1-type pSi-PNIPAM sample (Figure 4d). In Figure 4c, a reversible and fast (within 1 min) increase of about 1.21.3% in the ΔEOT% was observed upon changing the temperature from 25 to 40 °C. The sample remained stable and exhibited thermal switching for several cycles. A slight decrease in the EOT baseline was observed while the ΔEOT% remained virtually the same. This decreasing baseline is therefore attributed to degradation or further oxidation of the underlying pSi layer rather than to partial loss of the PNIPAM coating. In contrast to the positive switching observed on the P1-type pSi-PNIPAM sample, a slight decrease in ΔEOT% (approximately 0.3%) on heating was seen in the case of the control (Figure 4b). This decrease can be attributed to the expected small change of refractive index of water (of about 0.25%) on heating between 20 and 40 °C.65 Figure 4d shows the ΔEOT% recorded on an 7848
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Langmuir N1-type pSi-PNIPAM sample, measured on thermally cycling the hydrated sample above and below the LCST of PNIPAM. This sample showed an increase in ΔEOT% of around 2.6% on changing the temperature from 25 to 40 °C, which was about two times higher than the change measured on the P1-type samples. Since the polymer thickness was approximately the same for both samples (75.4 ( 2.3 nm for the P1 sample and 73.2 ( 4.6 nm for the N1 sample), this difference in ΔEOT% was attributed to the difference in pore size and/or porosity between the two samples. The effect of pore size on ΔEOT% will be addressed further below in the paper. It is useful to discuss the results shown in Figure 4bd in the context of the existing literature. Segal et al.15 performed EOT measurements on pSi samples impregnated with cross-linked PNIPAM hydrogels. These samples showed a decrease in EOT upon raising the temperature beyond the LCST of PNIPAM, and an increase in EOT when the sample was allowed to cool again. This effect is at odds with the well-known increase in refractive index of PNIPAM upon heating above the LCST.66 The authors explained the decrease on the basis that the hydrogel held above the LCST in the confined environment of the pores exerts a compressive stress on the pSi film, resulting in a decrease in the depth of the porous layer as the polymer chains collapse. The authors propose that this decrease would overshadow the increase in refractive index of the polymer upon collapse. We believe that the conspicuous differences between our results and the results of Segal et al.15 can be easily reconciled when considering the different polymer architectures used, that is, thin grafted polymer coatings versus a cross-linked bulk hydrogel. The absence of a cross-linker in our system ensures that the polymer chains collapse more or less independently of each other when heated above the LCST. Consequently, little or no strain will be exerted on the pSi film as the polymer collapses. As a result, the EOT change will be completely governed by the change in refractive index of the polymer, resulting in a net increase in EOT upon heating. Segal et al.15 reported changes in ΔEOT% magnitudes ranging from 0.1 to 4%. Interestingly, despite the opposite switching behavior, these values compare closely to our investigation where we observed ΔEOT% changes ranging from 0.3 to 3.5%. Influence of PNIPAM Film Thickness on Thermoresponsiveness. The relation between the EOT change and the PNIPAM film thickness was studied using N1-type pSi films. Using the “grafting from” approach, we could tune PNIPAM film thicknesses on a pSi-PNIPAM sample with 120 ( 53 nm pore size by simply varying the polymerization time from 0 to 15 min while using the same concentrations of monomer, ATRP catalyst, and ligand. Increased polymerization times led to increased length and molecular weight of the PNIPAM chains,39 and subsequently increased coating thickness as determined by ellipsometry (Table 3). For the 0 min sample, a PNIPAM thickness of 0 nm was assumed. The ΔEOT% when the sample was heated or cooled between 25 and 40 °C was then determined from the EOT sensorgram of the pSi-PNIPAM samples. The results presented in Table 3 show that ΔEOT% increases with dry film thickness. Interestingly, the PNIPAM film of less than 40 nm thickness does not show a positive change in the EOT when elevating the temperature above the LCST. On the contrary, this sample shows a slight decrease in EOT similar to the one observed in Figure 4b for the BiBAPTES functionalized sample without PNIPAM. It is conceivable and consistent with the literature
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Table 3. Change in ΔEOT% with Dry Film Thickness (Measured by Ellipsometry) on N1-type pSi-PNIPAM Samples polymerization time (min) 0
film thickness (nm) 0.0
ΔEOT% 0.22 (0.03
2
39.8 ( 5.8
3
47.3 ( 5.9
1.16 (0.80
5
56.5 ( 1.3
1.84 (0.80
10 15
72.1 ( 3.7 108.6 ( 5.0
2.52 (0.21 6.24 (1.23
1.04 (0.29
that PNIPAM based polymers of low molecular weight do not show a pronounced volume-phase transition.29,33 As stated in the previous section, the main reason for the increase in the EOT exhibited by the samples on temperature cycling across the LCST is the change in refractive index of PNIPAM upon collapse. At temperatures below the LCST of PNIPAM, the polymer is expected to be well hydrated and so the refractive index of the film should approach that of water (n = 1.33).65 Furthermore, the refractive index of dehydrated PNIPAM has been reported to be 1.508.66 Hence, a maximum ΔEOT% approaching 13% can be expected upon LCST switching. However, it is not expected that all the water in the PNIPAM film will be completely expelled upon collapse, as it has been shown that a significant amount of water is retained in the collapsed state of PNIPAM (above LCST).53 As a consequence, the apparent refractive index change of the pSi-PNIPAM hybrid is expected to be lower than 13%. The increase in ΔEOT% with increasing PNIPAM film thickness is easily explained by the fact that thicker PNIPAM films will expel larger volumes of water out of the pores upon collapse and hence a greater change in apparent refractive index will be evidenced. Laterally Graded pSi-PNIPAM Films. Experiments to investigate the effect of varying pore size of the pSi layer on the change in EOT were performed using lateral pSi gradients prepared following a procedure described in the literature.67 The advantage of utilizing the gradient format is that a spectrum of pore sizes is displayed on a single specimen. In our case, we produced a gradient with pore sizes ranging from a few hundred nanometers close to the position of the cathode during etching down to less than 20 nm at a position furthest away from where the cathode was placed during etching (Figure 5 and Table 1). Porosity decreases in the same direction. Polymer films of around 51.4 ( 3.8 nm dry thickness (determined by ellipsometry on flat Si) were grafted from laterally graded pSi (PGRAD in Table 1). and IRS readings were taken on seven spots along the sample at 1 mm intervals. Figure 6 illustrates the relation between ΔEOT% and the average pore size measured at each region on the gradient pSi-PNIPAM samples. Figure 6 also shows that the thickness of the pSi film decreased with increasing distances from the cathode during etching, as expected. The latter effect is decoupled from the changes in ΔEOT%, since these results were normalized by the EOT at 25 °C (below the LCST of PNIPAM) obtained on the respective spots. We observed that the ΔEOT% values are dependent on pore size, where for macroporous regions we observed values of up to 2.4%, while ΔEOT% approaches 0.3% for pore sizes below 25 nm. These results can be easily reconciled in terms of a decrease in polymer loading with decreased pore size and porosity along the 7849
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Langmuir
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Figure 5. Photograph of a pSi gradient film (PGRAD in Table 1) and SEM images showing change in pore sizes along seven different regions of the gradient. Scale bar on the SEM images is 200 nm and on the photograph 0.5 cm.
gradient. We note that the ΔEOT% also increased in the study by Segal et al.,15 for discrete samples of increasing pore size and porosity. Controlled Drug Release from pSi-PNIPAM. The model drug used for these experiments was camptothecin, a fluorescent anticancer drug.68 We found that optimal loading into the pSiPNIPAM (P1 and P2 types) was achieved in DMFwater mixtures displaying cononsolvency behavior toward PNIPAM.69 Kinetic experiments were conducted in order to monitor the release of camptothecin from the P1-type pSi-PNIPAM samples over time. As a negative control, we used BiBAPTES samples loaded under the same conditions as the pSi-PNIPAM samples. We also confirmed that the fluorescence of camptothecin in PBS buffer was stable over a 24 h period. In order to calculate the amount of camptothecin released over time, a calibration curve was constructed by measuring the fluorescence intensities of 1 nM to 6 μM dilutions of camptothecin in PBS. The data obtained from the release experiments were then fitted to the calibration curve, and the rate of release was calculated (Figure 7). After 300 min, the pSi-PNIPAM sample (Figure 7a) released significantly higher amounts (>0.3 nmol mm2) of the drug as compared to the control sample (Figure 7b) (
