ARTICLE pubs.acs.org/Langmuir
Dual Responsive Nanostructured Surfaces for Biomedical Applications Gabriela V. Martins,†,‡ Jo~ao F. Mano,†,‡ and Natalia M. Alves*,†,‡ †
3B's Research Group Biomaterials, Biodegradables, and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, S. Claudio do Barco, 4806-909 Caldas das Taipas Guimar~aes, Portugal ‡ IBB Institute for Biotechnology and Bioengineering, PT Government Associated Laboratory Braga, Portugal ABSTRACT: In this paper, we describe the construction and characteristics of thermoresponsive, thin nanostructured films prepared by layer-by-layer sequential assembly of chitosan-graft-NIPAAm and alginate. FTIR and 1H NMR spectra have confirmed the introduction of NIPAAm moieties onto the chitosan backbone. The LCST of the synthesized copolymer was found to be around 3133 °C. The formation of the polyelectrolyte multilayers containing the copolymer and alginate was followed in situ by quartz crystal microbalance with dissipation monitoring technique and ex situ by UVvis measurements. Our results revealed the linear increase of the multilayer film growth and the influence of the presence of salt. Moreover, AFM analysis has confirmed that PNIPAAm is able to reconform upon temperature swaps even when combined with other layers in a polyelectrolyte multilayer, demonstrating that the nanoassemblies are thermoresponsive. Preliminary results showed that, upon reducing culture temperature below PNIPAAm LCST, a gradual detachment of cell sheets from these PNIPAAm-based coatings has occurred.
’ INTRODUCTION “Smart” or stimuli-responsive polymers react to small shifts in their environment with dramatic changes in their physical properties.1 Most of the work has been dealing with temperature, pH, and ionic strength stimuli, due to the importance of these variables in typical physiological, biological, and chemical systems.2 “Smart” polymers undergo reversible phase transition involving mainly changes in their solubility, and ideally, this response should also be reversible. Nowadays, these thermosensitive polymers are playing an important role as self-regulated drug delivery systems3 in the separation and purification of biological materials,4 as injectable hydrogels for local wound repair58 and more recently for cell sheet engineering.9 One of the major advances in tissue engineering has been the use of sheets of cells for tissue reconstruction instead of individual cells. Cell sheet engineering is based on the control of cellular adhesion in a way that intact sheets of confluent cells can be recovered while keeping their morphology and function.10 Poly(N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive polymer with a lower critical solution temperature (LCST) of 3233 °C,11 enclosing both hydrophobic and hydrophilic domains below and above the LCST.12 At temperatures below the LCST, the polymer is soluble in aqueous solution, preferring an expanded or hydrated coil conformation. Above this temperature, the polymer becomes insoluble, preferring a folded globular structure. These temperature-dependent interactions arise from the balance between hydrogen bonding of hydrophilic segments of the polymer chain and hydrophobic interactions among hydrophobic isopropyl domains.12,13 The phase transition occurs over a narrow temperature range and is reversible by lowering the temperature. The thermoresponsive r 2011 American Chemical Society
behavior of PNIPAAm provides a convenient handle for changing the properties of biomaterial surfaces and their subsequent application as switchable or tunable membranes, coatings, or adsorbents.14,15 For biomedical applications, biocompatibility and biodegradability are indispensable requirements for successful outcome. Thus, one major disadvantage of the PNIPAAm polymer is its non-biodegradability.16 Therefore, to pursue partial biodegradability, this material is often combined with biopolymers. A novel water-soluble and thermosensitive PNIPAAm-grafted chitosan copolymer has been prepared for drug- and cell-delivery applications showing no or little cytotoxicity.17 Additionally, the graft of small chains of NIPAAm onto chitosan polymer backbone enables the development of materials exhibiting both temperature and pH dependence. Chitosan has pH-sensitive properties due to the protonationdeprotonation equilibrium of the amino groups and displays reversible conformational transitions in response to the net charge. Nanoparticles prepared from chitosan and PNIPAAm polymers were found to be environmentally responsive, and their particle size could be precisely manipulated by changing the pH value and temperature of the surrounding medium.18 A facile and controllable approach to attach these polymers to surfaces is using a layer-by-layer (LbL) technique. This method of surface engineering involves the formation of polyelectrolyte multilayers by sequentially treating a charged surface with solutions of oppositely charged polyelectrolytes.1922 The main Received: March 4, 2011 Revised: May 15, 2011 Published: June 03, 2011 8415
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Langmuir advantage of electrostatic LbL assembly is that it can be used to coat a wide range of complex substrates, so the control of the wettability by changing the temperature has applications in a broad range of fields such as thermoresponsive microcapsules,23 smart polymer nanocontainers,24 or thermally responsive filters for oil/water separation.25 Since the phase transition of PNIPAAm occurs at ranges close to the body temperature, chitosangraft-NIPAAm materials can be potentially used in pharmaceutical and medical applications. A promising and new cell recovery technology using nanometer-thick thermoresponsive PNIPAAm-based surfaces has emerged. Cells cultured on PNIPAAm surfaces at 37 °C (above the LCST) were found to detach spontaneously by lowering the medium temperature (below the LCST) and changing the hydration of the PNIPAAm chains.26 Moreover, the detached cells maintained their associated state and could be subsequently recultured, transplanted, or used for differentiation protocols.27,28 In this study, we describe the in situ formation and characteristics of a new thermoresponsive thin film built by electrostatic assembly. The grafting of NIPAAm into chitosan enables the design of “smart” polymers that mimic the reaction of biopolymers. The aim of this research is to investigate the thermosensitive behavior of the nanostructured assembly films. The technological relevance of the present work is due to the fact that PNIPAAm LCST lies between room temperature and body temperature, and so, a high interest in biological applications can be foreseen. The LbL approach described here presents a potential alternative to the traditional grafting techniques employed for cell sheet purposes. Polyelectrolyte-based substrates can gently detach cell sheets with their associated extracellular matrix and, thus, can undergo important implications for tissue engineering and regenerative medicine.10,29 The mild conditions and the absence of harmful solvents, the simplicity of the method, and the ability to modulate cellular adhesiveness by controlling the (nanometer) thickness of the coatings are some of the advantages of this novel strategy. In fact, assembling an LbL film onto a culture dish is much more simple and facile than performing the PNIPAAm grafting onto this substrate. Moreover, LbL thermosensitive constructions allow precise control over the layer thickness on the order of 12 nm, which is a determinant factor for developing films suitable for cell sheet engineering.30,31
’ MATERIALS AND METHODS Materials. Chitosan (CHI; medium molecular weight, pka ≈ 632) was purchased from Sigma-Aldrich and purified prior to use by recrystallization. The degree of deacetylation (DDA) of chitosan, 7880%, was determined by proton nuclear magnetic resonance spectroscopy (1H NMR).33 Sodium alginate derived from brown algae (ALG; low viscosity, pka ≈ 3.4 and 3.634) was obtained from Sigma and used as received. N-Isopropylacrylamide (NIPAAm; Acros) was purified by recrystallization from n-hexane/diethyl ether mixture (5:1, v/v) and dried in vacuo for 24 h. Ceric ammonium nitrate (CAN; Sigma-Aldrich) was used as received without further purification.
Preparation and Characterization of Chitosan-graft-NIPAAm. Graft polymerization of NIPAAm onto chitosan was carried out using CAN as an initiator. Chitosan was dissolved in 5% (w/w) aqueous acetic acid solution. While bubbling nitrogen gas, NIPAAm monomer and 5 103 M of CAN were successively added into the chitosan solution and the reaction mixture was stirred at room temperature for 30 min. Afterward, the reaction was continued at 30 °C for
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16 h. After graft polymerization, the product solution was concentrated on a rotary evaporator and then precipitated in excess acetone and separated by filtration. The resulting product was dissolved in distilled water and dialyzed (cellulose membranes, Sigma-Aldrich) at 4 °C during 1 week. The chemical structure of the chitosan-graft-NIPAAm product was investigated by Fourier transform infrared (FTIR) and 1H NMR spectroscopy techniques. FTIR spectra were recorded on an IR Prestige-21 (Shimadzu) spectrometer and samples were prepared in KBr pellets. 1H NMR spectra were obtained from a Varian Unity Plus 300 spectrometer (at 300 MHz) using 1 vol % acetic-d3 acid in D2O as solvent.
Thermoreversible Behavior of Chitosan-graft-NIPAAm. The turbidity of the polymeric solutions was determined by measuring the absorbance at 400 nm in a Multi-Detection Microplate Reader Synergie HT (BIO-TEK) UVvisible spectrophotometer. The temperature was raised from 28 to 38 °C in 1 °C increments with 10 min of incubation per increment. Solutions were prepared using ultrapure water with and without NaCl salt (0.15 M). The cloud point temperature was defined as the temperature at the inflection point in the absorbance versus temperature curve. The hydrodynamic diameter (Dh) and size distribution of chitosangraft-NIPAAm in solution was determined by dynamic light scattering (DLS) experiments performed with a Zetasizer NanoZS Instrument (ZEN3600, Malvern Instruments). The measurements were conducted in the temperature range 2040 °C with a temperature interval of 1 °C. Solutions with and without NaCl salt (0.15 M) were prepared in ultrapure water and carefully filtered using a 0.22 μm disposable membrane filter.
Buildup and Characterization of PNIPAAm-Based Films. Fresh solutions of chitosan-graft-NIPAAm (positively charged polymer) and alginate (negatively charged polymer) were prepared in aqueous medium and in saline conditions (0.15 M NaCl) to yield a final polymer concentration of 0.5 mg/mL. The pH of the solutions was adjusted with acetic acid and sodium hydroxide solutions to a final value of 5.5. The multilayer formation was followed in situ by quartz crystal microbalance with dissipation monitoring (Q-Sense, E4 system). QCM-D has the ability to simultaneously measure the normalized resonant frequency (Δf/n) and energy dissipation (ΔD) shifts. This technique has been extensively described in detail elsewhere.35 Very briefly, AT-cut quartz crystals with gold-plated polished electrodes (Q-Sense) can be excited at its fundamental frequency (f0 ≈ 5 MHz) as well as at the third, fifth, seventh, ninth, and eleventh overtones, corresponding to 15, 25, 35, 45, and 55 MHz, respectively. Before use, gold-coated crystals were cleaned with sequential sonication in water, ethanol, and water and then dried with flowing nitrogen gas. QCM-D experiments were started with a buffer baseline, ultrapure water, or 0.15 M NaCl solution. Then, the multilayer films were built by alternating chitosan-graft-NIPAAm and alginate depositions onto the quartz crystal. The gold substrates were first coated with the polycation layer (chitosan-graft-NIPAAm). Fresh polyelectrolyte solutions were injected into the measurement chamber for 10 min at a flow rate of 100 μL/min, and a washing step of 5 min with the respective buffer solution was included after the adsorption of each polyelectrolyte. During the whole process, Δf/n and ΔD variations were continuously recorded as a function of time. For all measurements, the temperature was set up at 25 °C. The experiments were performed at least in triplicate. Afterward, to study the thermoresponsive properties of the adsorbed polymers, the coated quartz crystals were subjected to a gradual increase of temperature until 38 °C and continuously followed by means of QCM-D. The interpretation of the viscoelastic properties of PNIPAAm-based films was assessed with a Voigt-based model36 that consists of a parallel combination of a spring and dashpot to represent the elastic (storage) 8416
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Langmuir and inelastic (damping) behavior of a material, respectively. The QTools software (v 3.0.6.213) provided with the equipment (Q-Sense) was used to model our QCM-D data according to the above-mentioned Voigt model. Δf/n and ΔD shifts were fitted for three overtones (7th, 9th, and 11th). The thickness of the wet films was calculated by fitting in an iterative fashion and by assuming a fluid density of 1000 kg/m3, a fluid viscosity of 0.0012 kg/ms, and a layer density of 1100 kg/m3. For UVvisible spectral and atomic force microscopy (AFM) measurements, a chitosan-graft-NIPAAm/alginate multilayered system was prepared on glass coverslips (ø = 13 mm, Agar Scientific) by manual dipping. Glass substrates were preliminary cleaned with ethanol, extensively rinsed with water, and dried under nitrogen. The process of selfassembly was performed in the same conditions described above (solution concentration, pH, temperature, and deposition time were the same as those used for QCM-D experiments). UVvis spectra were recorded on a UVvis 2401 (Shimadzu) spectrophotometer, and to minimize background interferences, no additional salt was added to any of the polymer or rinse solutions. AFM imaging was performed with an Agilent Technologies 5500 coupled with a temperature controller. The measurements were conducted on an area of 5 5 μm2 by immersing the tip assembly in ultrapure water at 23 and 40 °C. Images were scanned in tapping mode using silicon cantilevers with a resonance frequency of 230 kHz (RTESP, Veeco). Before each measurement, samples were preincubated in situ in the liquid for 20 min.
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Figure 1. Chitosan-graft-NIPAAm solution (A) below LCST and (B) above LCST.
Cell Culture and Temperature-Induced Detachment of Cell Sheets. For the cellular study, glass coverslips (ø = 13 mm) coated with chitosan-graft-NIPAAm/alginate multilayer films were prepared in a homemade automatic dipping machine. These modified substrates were placed into 24-well tissue culture plates (nonadherents) and then sterilized by UV-radiation during 15 min. A human osteoblast-like cell line (SaOs-2; European Collection of Cell Cultures, UK) was cultured in a humid 37 °C/5% CO2 incubator in pH ≈ 7.4 growth media consisting of Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% of heat-inactivated fetal bovine serum (FBS; Biochrom AG, Germany) and 1% antibioticantimicotic (Gibco, USA). The sterile samples were prewashed with phosphate buffered saline solution (PBS; Sigma) and then seeded with the SaOs-2 cell line to a final density of 3.3 104 cells per mL (1.5 mL per well). Cells were cultured onto the multilayer films at 37 °C, 5% CO2 in a humidified atmosphere, and growth medium was renewed every 3 days. After reaching confluence (∼6 days), cell detachment from the PNIPAAm-based films was performed by incubating the culture plate at 4 °C for short periods of time (10 and 15 min). The detachment of the cultured SaOs-2 cell sheets on the coated substrates was examined with Inverted Microscope Zeiss Axiovert (40 PG-HITEC). To evaluate cellular behavior after cooling protocol, detached cell sheets were recultured on adherent tissue culture plates.
Figure 2. FTIR spectra of (a) chitosan, (b) PNIPAAm homopolymer, and (c) chitosan-graft-NIPAAm.
’ RESULTS AND DISCUSSION Preparation and Characterization of Chitosan-graft-NIPAAm (GRAFT). The final product obtained was soluble in
slightly acidic aqueous media at low temperatures, showing a transparent liquid appearance. It was observed that, when temperature is increased above the lower critical solution temperature (LCST), solution changed to a white opaque aspect (Figure 1). Although the homopolymer PNIPAAm is completely soluble in water at temperatures below 3233 °C, our final product contains chitosan, hindering their complete solubility in water. The analysis of FTIR (Figure 2) and 1H NMR (Figure 3) data showed that CAN initiator was successfully used to introduce the
Figure 3. 1H NMR spectra of (a) chitosan, (b) PNIPAAm homopolymer, and (c) chitosan-graft-NIPAAm.
NIPAAm monomer onto the chitosan polymer backbone. The results obtained seem to be in agreement with previous work performed by Kim et al.,37 Seetapan et al.,38 and Lee et al.39 Figure 2 presents the FTIR spectra of PNIPAAm homopolymer, chitosan-graft-NIPAAm, and chitosan. A peak can be observed in the spectrum of the grafted product at around 1120 cm1 that could be assigned to the CO stretching vibration of chitosan. 8417
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Figure 4. Turbidity curves of PNIPAAm homopolymer and chitosangraft-NIPAAm as function of temperature (0.5 mg/mL polymer concentration in water and NaCl 0.15 M media).
Characteristic absorption bands of PNIPAAm homopolymer, such as the amide I and II bands at 1655 and 1540 cm1, respectively, are also strengthened in the grafted product (Figure 2b). 1H NMR spectra also confirm the grafting of NIPAAm onto the chitosan chain. Signals at 1.14, 1.58, and 1.70 ppm, in the spectrum of the grafted material (Figure 3c), corresponding to protons in the NIPAAm moiety are an indication that the introduction of NIPAAm units onto the chitosan chain occurred without a clear modification of its NMR spectrum. Moreover, the spectrum of chitosan (Figure 3a) exhibits the typical peaks, including the proton on the carbon bearing the amine groups (at 3.13 ppm). Although with a slight shift to the right side, this characteristic peak of chitosan was also found in the spectrum of the grafted product. Thermoreversible Behavior of Chitosan-graft-NIPAAm. Figure 4 shows typical absorbance versus temperature curves for the grafted product and PNIPAAm homopolymer, with and without NaCl salt. Although it has been widely presented that the inclusion of comonomers enables some tuning of the phase transition of PNIPAAmr,4044 no significant variation was observed (just ∼1 °C difference). Like expected, solutions of PNIPAAm homopolymer displayed a phase transition temperature around 33 °C in aqueous conditions,45 while the grafted polymer showed the respective transition at 34 °C. It was thought that the cloud point of chitosan-graft-NIPAAm would increase, by comparison with the PNIPAAm homopolymer, due to the hydrophilic character of the protonated NH3þ groups of chitosan units. Nevertheless, data seem to indicate that the balance between hydrophilicity and hydrophobicity in the polymeric structure was not significantly affected. The reason for this outcome can be the fact that PNIPAAm moieties were incorporated as pendant chains into the backbone of chitosan, and by being so, the conformational transition from coil to globule state was not influenced.4 Moreover, the determination of the cloud point was found to occur in a sharp and narrow range. In parallel, Figure 4 also indicates that the macroscopic phase separation of the side chains of PNIPAAm is less pronounced for the grafted product due to the presence of chitosan, a non-thermoresponsive polymer. Despite the fact that the determination of the cloud point has been accepted as the common way to estimate the LCST of thermoresponsive polymers in aqueous solutions, we should be aware that it is indeed an overestimation with some limitations.46
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Figure 5. Temperature dependence of the hydrodynamic diameter (Dh) of chitosan-graft-NIPAAm product in aqueous solution, with and without NaCl salt.
Thus, the LCST behavior of chitosan-graft-NIPAAm solutions was also characterized by dynamic light scattering (DLS), as seen in Figure 5. Our results corroborate earlier statements that nanosized aggregates are observed by DLS at lower temperatures than the cloud point temperature determined by turbidimetry.46 For this thermosensitive graft polymer, LCST values were found to be 2 °C lower compared with the respective cloud points. Concerning the effect of the presence of NaCl salts in the background solution, the results are in agreement with the general principle that the LCST may be decreased (known as “salting-out”) depending on the chemical structure of the added salt.13,47 The simple explanation for this effect arises from the fact that, during the dissolution of thermoresponsive polymers, water molecules will also interact favorably with the charged entities of the buffer salts in the solution, and so, competition reactions will take place.48 Although in this case the influence of NaCl in the graft product LCST is rather small (∼2 °C), our data supported the supposition that the presence of the salts in buffer solutions tends to reduce the phase transition of PNIPAAm. Buildup and Characterization of PNIPAAm-Based Films. Figure 6 compares the build-up of chitosan-graft-NIPAAm and alginate multilayered assemblies with (A) and without (B) NaCl. Although we were able to build NIPAAm-based films for both conditions, their growing behavior was slightly different. For an ionic strength of 0.15 M (Figure 6A), the decrease of Δf/n after each polymer adsorption evidence that mass is being deposited regularly at the crystal surface. In the absence of NaCl (Figure 6B), film formation was more modest, especially concerning alginate layers that have displayed low Δf/n variations. Even so, the adsorption was still sufficient for the graft polymer to be adsorbed in the subsequent adsorption step. Our results are in agreement with the general rule that, without salts, the polyelectrolyte chains adopt a more extended conformation containing fewer loops and tails, and thus, the polymer adsorbs as thinner layers.20,49 Still, the sequential deposition of these nanosystems was generally stable and reproducible. Furthermore QCM-D is sensitive to any adsorbed mass associated with the films, including solvent. QCM-D technology has already been acknowledged as a useful technique to follow the hydration of thermosensitive polymer brushes.50 Herein, data have shown that the obtained multilayers present a pronounced viscoelastic behavior, as each polymer deposition is accompanied by an increase of ΔD. In 8418
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Figure 8. UVvis spectra of the sequential adsorption of (GRAFT/ ALG)5 multilayer films in aqueous conditions, for n = 1, 3, 5, 7, 9 (n means number of layers of GRAFT/ALG films).
Figure 6. QCM-D results showing the in situ buildup of (GRAFT/ ALG)5GRAFT films at a pH of 5.5 with (A) and without (B) NaCl. Steps of 10 min were taken for polymer adsorption and 5 min for an afterward rinsing. Normalized frequency (Δf/n) and dissipation (ΔD) changes were recorded as a function of time. Plots represent 7th Δf/n (9), 9th Δf/n (b), 11th Δf/n (2), 7th ΔD (0), 9th ΔD (O), and 11th ΔD (Δ).
Figure 7. Frequency (Δf/n) (squares) and dissipation (ΔD) (circles) changes as a function of the number of layers for (GRAFT/ALG)5GRAFT films, for the 9th harmonic. The deposition was carried out at pH of 5.5 and with an effective ionic strength of 0.15 M with NaCl, both having polyelectrolyte concentration 0.5 mg/mL.
addition, Figure 7 displays the frequency and dissipation changes as a function of the number of layers for (GRAFT/ALG)5GRAFT
films in the presence of NaCl salt. In accordance with previous layer-by-layer (LbL) studies containing chitosan and alginate polyelectrolytes,51,52 our results indicated that the multilayer growth occurs in a linear regime. Although data are not presented here, a final thickness of 4550 nm was achieved for the (GRAFT/ALG)5GRAFT assembly in salt conditions, in the wet state (solvent included). It was found that the film thickness increases linearly as the number of layers increased, at a rate of ca. 4 nm per layer. Hence, the average layer thickness of the GRAFT/ ALG multilayers was apparently thicker than that of the CHI/ALG multilayers (3 nm).53 The obvious explanation for this outcome can be the introduction of the pendant PNIPAAm chains that with their stretched conformation will contribute to the increase of the molecular weight, leading to thicker multilayers.54 The buildup of multilayers from chitosan-graft-NIPAAm and alginate aqueous solutions was also monitored using UVvis technology (Figure 8). The spectra of the PNIPAAm-based films presented a maximum absorbance around 290 nm, which could be ascribed to the CdO groups of chitosan. Since the terminal layer can have a great impact on the overall properties/behavior of the Lbl nanoassemblies, the measurements were exclusively conducted for films ending with a chitosan-graft-NIPAAm layer. Although the intensity of the absorbance increased with the growth of (GRAFT/ALG)5 films, the buildup tendency was not linear, but interestingly, two different regimes were found to describe the formation of the responsive layers. The first few layers can be assigned to the adsorption and reorganization of the polyelectrolyte at the solid substrate surface (glass coverslips) that may not be as uniformly charged. Thus, regarding the influence of the substrate that holds the film growth, earlier investigations are in agreement with our findings that, until 5 to 6 deposition cycles, the polyelectrolyte assemblies can still “feel” the influence of the underneath supporting material.20,55 Moreover, there is evidence that for values of absorbance above 2 the BeerLambert law can suffer some deviations,56 and so, the final impact of this result should hold mainly a qualitative significance. We showed that the synthesized grafted product is thermoresponsive in bulk solutions and that it was possible to produce multilayers containing PNIPAAm. It would be interesting to investigate if thermoresponsiveness is maintained within the 8419
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Figure 9. Temperature dependence of dissipation (ΔD) variations for (GRAFT/ALG)5GRAFT films assembled in the same conditions as described for Figure 6. Gray area concerns the region where the LCST value of the thermoresponsive polymer was found.
multilayered architecture. In order to study the thermoresponsive character of the adsorbed polymers, the (GRAFT/ALG)5GRAFT films coated on the quartz crystals were subjected to a gradual increase of temperature in the QCM-D and the change in dissipation was studied as a function of temperature. Figure 9 clearly shows a two-regime behavior as a result of the discontinuous character of ΔD near the LCST value. Data seem to suggest that the gray area points out the transformation from a random coil to a dense globular structure. Here, all the materials had a specific response to temperature variation, but the change in the slope for a steeper one constitutes an indication that PNIPAAm extended chains are collapsing and expelling molecules of water.57 After evaluating the thermoresponsiveness behavior of the PNIPAAmbased films during a heating process, the temperature was decreased to 25 °C to assess if the response was reversible. The dissipation returned to the initial value (data not showed) indicating that the process was completely reversible on the surface. The results demonstrated that PNIPAAm segments are able to reconform even when combined with other layers in a polyelectrolyte multilayer and, thereby, that the nanoassemblies are thermoresponsive. Although a recent work characterized the thermoresponsivity of adsorbed layers,58 to our knowledge, this is the first attempt to determine the LCST of PNIPAAm polymer enclosed in a multilayered assembly using QCM-D measurements. In order to further characterize the adsorbed layers, AFM images were collected for (GRAFT/ALG)5GRAFT films, and the results are shown in Figure 10. The presented data display the morphological differences on the surface of PNIPAAm-based films as a response to an increase of temperature above the LCST value. The thermoresponsive layers at room temperature exhibit a rough surface, with a root-mean-squared roughness (rms) value of 44.5 nm. In contrast, the surface of these assemblies becomes quite smooth, with a rms of 18.3 nm, when the temperature is increased for 40 °C. Moreover, above LCST, AFM imaging seem to show the aggregation structure formed from PNIPAAm folded globule conformation.43,59 These results are in agreement with earlier findings claiming that the surface morphology gets smoother with increasing temperature.15 Additionally, LbL self-assemblies containing PNIPAAm have also confirmed that at higher temperatures this thermosensitive polymer adopts a tighter coil structure, leading to tighter packing and thereby to a smoother film.60
Figure 10. Surface topography images of (GRAFT/ALG)5GRAFT films in hydrated state at 23 °C (A) and at 40 °C (B). The images were taken by AFM in tapping mode (5 5 μm2) and the root-mean-squared roughness (rms) values are also displayed.
Cell Culture and Temperature-Induced Detachment of Cell Sheets. The biocompatibility and thermoresponsiveness
character of the self-assembled multilayers were assessed by examining cellular morphology, adhesion, and proliferation. The SaOs-2 cells cultured on the (GRAFT/ALG)5GRAFT coated substrates showed good adhesion behavior followed by a normal growth and, similar to our previous studies with chitosanalginate films,52 confluence was reached after less than 1 week. Figure 11A,B presents the detachment of the cell film from (GRAFT/ALG)5GRAFT coated surfaces after incubation at 4 °C for 10 and 15 mim, respectively. It was observed that cell sheet detachment from these PNIPAAm-based surfaces occurred gradually from the sheet periphery toward the interior. Moreover, during the detachment period, the detached cells remained spread. Thus, upon reducing culture temperature below PNIPAAm LCST, culture surfaces become hydrophilic and the adhered cells spontaneously detach, along with their deposited extracellular matrix (ECM). Such results demonstrated that these smart multilayered films can be used to control cell attachment. However, in our case-study, we were not able to obtain one single-celled sheet for all the extension of the coated substrate, but instead, Figure 11B displays the detachment of 8420
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Figure 11. In situ detachment of SaOs-2 cell sheets after 10 min (A) and 15 min (B) of incubation at 4 °C from (GRAFT/ALG)5GRAFT films. Microscope images of a detached cell sheet recultured in a new tissue culture plate on time 0 (C) and after 45 min (D).
cells on 1/3 of the surface. This outcome is not completely unexpected, since hydration of PNIPAAm at the cellmaterial interface is not the only factor that rules cell detachment from culture surfaces. We consider that there are many variables that can be still optimized in order to accelerate and extend the detachment process of the cell membranes, namely, the number of layers, the quantity of PNIPAAm grafted at the surface, or the nature of the polyanion.61,62 Additionally, it has already been acknowledged how the optimum temperature can be distinct for each type of cell as a response to the different metabolic requirements.26 Studies have also shown that cell sheets could resist detachment due to the ECM proteins that function as anchoring points and therefore remain attached to the surface after detachment procedure.63 Previous investigations based on contact angle measurements agree that the brush collapse is a more gradual transition than that observed in dilute aqueous solution.64 Furthermore, the behavior of polymer molecules when one of their ends is tethered to a surface or an interface is qualitatively different from that of chain molecules in bulk.65 Consequently, the degrees of freedom for PNIPAAm in a multilayer system will be substantially reduced as compared to solution.54 These important findings suggest that geometric constraints adversely affect the ability of polymer chains to adopt a more ordered and collapsed conformation. Similar to our findings, Jaber and Schlenoff have shown that the high degree of interpenetration present in a multilayer and the random nature of the copolymers can lead to a much lower extent of thermoresponsivity compared to what is observed in solution.66 As one of the functions of ECM is anchoring and providing support for cells, a method allowing cells to detach from their
culture surfaces with intact ECM is extremely desirable. Thus, the role of ECM proteins on cell behavior on temperature-responsive PNIPAAm films has been carefully analyzed. Previous results have shown that the LbL technique is an efficient approach to form PNIPAAm-based thermal responsive surfaces for cell growth and removal without enzymatic treatment.29 This gives the noninvasive harvest of cultured cells as an intact layer cell sheet containing deposited ECM. Unlike enzymatic dissociation, which breaks down the ECM proteins and the cell surface receptors, the intact cellECM communication in the thermo lift-off treatment is preserved.30 The detached cell sheet was transported to a new tissue culture plate for reculture in order to assess cellular behavior after the cooling protocol. As previously reported, we noted that, upon the detachment (Figure 11C), there is a tendency of the cells to clump up into cell clusters.67 After a short period of time (less than 1 h), we observed that the cell sheet started to spread in order to attach to the new surface (Figure 11D). These preliminary results constitute strong evidence that the metabolic behavior of the detached cells did not seem to be harmed during the detachment of the cell sheets. The present work has confirmed that nanoassemblies containing PNIPAAm moieties still keep temperature sensitivity that can be used to induce noninvasive recovery of culture cell-viable monolayers by simple temperature manipulation. Moreover, this new approach based on LbL technology enables a higher control concerning the thickness and structural arrangement of the multilayered coatings used for cell culture and subsequent transplantation. The facile incorporation of growth factors into the nanoassemblies can also become an important issue for in situ cellular differentiation. 8421
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’ CONCLUSIONS The formation of a new kind of biocompatible and thermosensitive surface based on LbL sequential assembly of chitosangraft-NIPAAm and alginate polymers was investigated. In this study, we reported the preparation of a PNIPAAm-based polyelectrolyte system exhibiting a thermoresponsive behavior with a LCST of 3133 °C. QCM-D measurements combined with UVvis analysis revealed the growth of the PNIPAAm-based multilayered system. Meanwhile, the morphology and responsive properties of the self-assembled film were evaluated by in situ temperature-controlled AFM. The thermosensitive behavior of the nanocoatings was confirmed by the transition from a globular to coiled structure of the polymer chains resulting in a more elastic surface for temperatures above the LCST. Moreover, our preliminary results have shown that (chitosan-graft-NIPAAm)/ alginate films enable the attachment and proliferation until confluence of cells at 37 °C and the spontaneous detachment of cell sheets along with their deposited extracellular matrix by simple temperature modulation. Thus, we have successfully designed thermoresponsive surfaces with nanostructure architectures that could have potential to be used in cell sheet engineering. Furthermore, the easy-to-build and the highly tunable features of these LbL coatings make them ideal candidates to be employed for drug delivery and controlled release systems, controllable membranes, filters, and sensory devices. ’ AUTHOR INFORMATION Corresponding Author
*Natalia M. Alves, Telephone: þ351 253510900, Fax: þ351 253510909, E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was partially supported by Portuguese Foundation for Science and Technology (FCT), through the PTDC/FIS/ 68209/2006 and PTDC/QUI/68804/2006 projects. The research leading to these results has also received funding for the European Union Seventh Framework Programme (FP7/20072013) under grant agreement no. NMP4-SL-2009-229292. ’ REFERENCES (1) Mano, J. F. Adv. Eng. Mater. 2008, 10 (6), 515–527. (2) Roy, I.; G., M. Chem. Biol. 2003, 10. (3) Qiu, Y.; Park, K. Adv. Drug Delivery Rev. 2001, 53 (3), 321– 339. (4) Zhang, H.-f.; Zhong, H.; Zhang, L.-l.; Chen, S.-b.; Zhao, Y.-j.; Zhu, Y.-l. Carbohydr. Polym. 2009, 77 (4), 785–790. (5) Van Tomme, S. R.; Storm, G.; Hennink, W. E. Int. J. Pharm. 2008, 355 (12), 1–18. (6) Baroli, B. J. Pharm. Sci. 2007, 96 (9), 2197–2223. (7) Mano, J. F.; Reis, R. L. J. Tiss. Eng. Regenerative Med. 2007, 1 (4), 261–273. (8) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32 (22), 7370–7379. (9) Tang, Z.; Akiyama, Y.; Yamato, M.; Okano, T. Biomaterials 2010, 31 (29), 7435–7443. (10) Weder, G.; Guillaume-Gentil, O.; Matthey, N.; Montagne, F.; Heinzelmann, H.; V€or€os, J.; Liley, M. Biomaterials 2010, 31 (25), 6436–6443. (11) Schild, H. G. Prog. Polym. Sci. 1992, 17 (2), 163–249. (12) Pelton, R. J. Colloid Interface Sci. 2010, 348 (2), 673–674.
ARTICLE
(13) Recillas, M.; Silva, L. L.; Peniche, C.; Goycoolea, F. M.; Rinaudo, M.; Arguielles-Monal, W. M. Biomacromolecules 2009, 10 (6), 1633–1641. (14) Rama Rao, G. V.; Krug, M. E.; Balamurugan, S.; Xu, H.; Xu, Q.; Lopez, G. P. Chem. Mater. 2002, 14 (12), 5075–5080. (15) Fujie, T.; Park, J. Y.; Murata, A.; Estillore, N. C.; Tria, M. C. R.; Takeoka, S.; Advincula, R. C. ACS Appl. Mater. Interfaces 2009, 1 (7), 1404–1413. (16) Seal, B. L.; Otero, T. C.; Panitch, A. Mater. Sci. Eng. R: Rep. 2001, 34 (45), 147–230. (17) Lee, J. W.; Jung, M. C.; Park, H. D.; Park, K. D.; Ryu, G. H. J. Biomater. Sci., Polym. Ed. 2004, 15, 1065–1079. (18) Chuang, C. Y.; Don, T. M.; Chiu, W. Y. J. Polym. Sci., Part A: Polym. Chem. 2010, 48 (11), 2377–2387. (19) v. Klitzing, R. Phys. Chem. Chem. Phys. 2006, 8 (43), 5012– 5033. (20) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21 (7), 319–348. (21) Boudou, T.; Crouzier, T.; Ren, K.; Blin, G.; Picart, C. Adv. Mater. 2010, 22 (4), 441–467. (22) Hammond, P. T. Adv. Mater. 2004, 16 (15), 1271–1293. (23) Glinel, K.; Sukhorukov, G. B.; M€ohwald, H.; Khrenov, V.; Tauer, K. Macromol. Chem. Phys. 2003, 204 (14), 1784–1790. (24) Gao, Y.; Chen, X.; Liao, B.; Ding, X.; Zheng, Z.; Cheng, X.; Pang, H.; Peng, Y. Polym. Bull. 2006, 56 (4), 305–311. (25) Kwak, D.; Han, J. T.; Lee, J. H.; Lim, H. S.; Lee, D. H.; Cho, K. Surf. Sci. 2008, 602 (19), 3100–3105. (26) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16 (4), 297–303. (27) Hatakeyama, H.; Kikuchi, A.; Yamato, M.; Okano, T. Biomaterials 2006, 27 (29), 5069–5078. (28) Nozaki, T.; Yamato, M.; Inuma, T.; Nishida, K.; Okano, T. J. Tiss. Eng. Regenerative Med. 2008, 2 (4), 190–195. (29) Liao, T.; Moussallem, M. D.; Kim, J.; Schlenoff, J. B.; Ma, T. Biotechnol. Prog. 2010, 26 (6), 1705–1713. (30) Kumashiro, Y.; Yamato, M.; Okano, T. Ann. Biomed. Eng. 2010, 38 (6), 1977–1988. (31) Fukumori, K.; Akiyama, Y.; Kumashiro, Y.; Kobayashi, J.; Yamato, M.; Sakai, K.; Okano, T. Macromol. Biosci. 2010, 10 (10), 1117–1129. (32) Rinaudo, M. Prog. Polym. Sci. 2006, 31 (7), 603–632. (33) Lavertu, M.; Xia, Z.; Serreqi, A. N.; Berrada, M.; Rodrigues, A.; Wang, D.; Buschmann, M. D.; Gupta, A. J. Pharm. Biomed. Anal. 2003, 32 (6), 1149–1158. (34) de Kerchove, A. J.; Elimelech, M. Macromolecules 2006, 39 (19), 6558–6564. (35) Marx, K. A. Biomacromolecules 2003, 4 (5), 1099–1120. (36) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59 (5), 391–396. (37) Kim, S. Y.; Cho, S. M.; Lee, Y. M.; Kim, S. J. J. Appl. Polym. Sci. 2000, 78 (7), 1381–1391. (38) Seetapan, N.; Mai-ngam, K.; Plucktaveesak, N.; Sirivat, A. Rheol. Acta 2006, 45 (6), 1011–1018. (39) Lee, J. W.; Jung, M. C.; Park, H. D.; Park, K. D.; Ryu, G. H. J. Biomater. Sci., Polym. Ed. 2004, 15, 1065–1079. (40) Jones, M. S. Eur. Polym. J. 1999, 35 (5), 795–801. (41) Eeckman, F.; Mo€es, A. J.; Amighi, K. Int. J. Pharm. 2004, 273 (12), 109–119. (42) Lee, B. H.; Vernon, B.; Situ-Gelling, In Macromol. Biosci. 2005, 5 (7), 629–635. (43) Bisht, H. S.; Wan, L.; Mao, G.; Oupicky, D. Polymer 2005, 46 (19), 7945–7952. (44) Serpe, M. J.; Jones, C. D.; Lyon, L. A. Langmuir 2003, 19 (21), 8759–8764. (45) Wu, C.; Zhou, S. Macromolecules 1995, 28 (24), 8381– 8387. (46) Lopez-Perez, P. M.; da Silva, R. M. P.; Pashkuleva, I.; Parra, F.; Reis, R. L.; San Roman, J. Langmuir 2009, 26 (8), 5934–5941. 8422
dx.doi.org/10.1021/la200832n |Langmuir 2011, 27, 8415–8423
Langmuir
ARTICLE
(47) Geever, L.; Nugent, M.; Higginbotham, C. J. Mater. Sci. 2007, 42 (23), 9845–9854. (48) Minoo, R. H.; Hassanzadeh, D.; Azarmi, S.; Entezami, A. A. Polym. Adv. Technol. 2007, 18 (12), 986–992. (49) Tjipto, E.; Quinn, J. F.; Caruso, F. Langmuir 2005, 21 (19), 8785–8792. (50) Annaka, M.; Yahiro, C.; Nagase, K.; Kikuchi, A.; Okano, T. Polymer 2007, 48 (19), 5713–5720. (51) Hillberg, A. L.; Holmes, C. A.; Tabrizian, M. Biomaterials 2009, 30 (27), 4463–4470. (52) Martins, G. V.; Merino, E. G.; Mano, J. F.; Alves, N. M. Macromol. Biosci. 2010, 10, 1444–1455. (53) Alves, N. M.; Picart, C.; Mano, J. F. Macromol. Biosci. 2009, 9 (8), 776–785. (54) Wang, F.; Li, L.; Gao, C. Macromol. Chem. Phys. 2009, 210 (23), 2022–2028. (55) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. Langmuir 1999, 16 (3), 1249–1255. (56) Song, Z.; Yin, J.; Luo, K.; Zheng, Y.; Yang, Y.; Li, Q.; Yan, S.; Chen, X. Macromol. Biosci. 2009, 9 (3), 268–278. (57) Wu, K.; Wu, B.; Wang, P.; Hou, Y.; Zhang, G.; Zhu, D.-M. J. Phys. Chem. B 2007, 111 (30), 8723–8727. (58) Utsel, S.; Malmstrom, E. E.; Carlmark, A.; Wagberg, L. Soft Matter 2010, 6 (2), 342–352. (59) Zareie, H. M.; Volga Bulmus, E.; Gunning, A. P.; Hoffman, A. S.; Piskin, E.; Morris, V. J. Polymer 2000, 41 (18), 6723–6727. (60) Quinn, J. F.; Caruso, F. Langmuir 2003, 20 (1), 20–22. (61) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20 (13), 5506–5511. (62) Cole, M. A.; Voelcker, N. H.; Thissen, H.; Griesser, H. J. Biomaterials 2009, 30 (9), 1827–1850. (63) da Silva, R. M. P.; Mano, J. F.; Reis, R. L. Trends Biotechnol. 2007, 25 (12), 577–583. (64) Fulghum, T. M.; Estillore, N. C.; Vo, C.-D.; Armes, S. P.; Advincula, R. C. Macromolecules 2007, 41 (2), 429–435. (65) Liu, G.; Zhang, G. J. Phys. Chem. B 2004, 109 (2), 743–747. (66) Jaber, J. A.; Schlenoff, J. B. Macromolecules 2005, 38 (4), 1300–1306. (67) Loh, X. J.; Cheong, W. C. D.; Li, J.; Ito, Y. Soft Matter 2009, 5 (15), 2937–2946.
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dx.doi.org/10.1021/la200832n |Langmuir 2011, 27, 8415–8423
