Controlled Assembly and Release of Retinoic Acid Based on the

Jan 16, 2013 - In this Article, we focus on the incorporation of RA, as a negatively charged moiety, into layered polyelectrolyte films on surfaces by...
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Controlled Assembly and Release of Retinoic Acid Based on the Layer-by-Layer Method Yueyue Chen,† Guanghong Zeng,‡ Fei Pan,† Junbo Wang,§ and Lifeng Chi*,† †

Physikalisches Institut and Center for Nanotechnology (CeNTech), Universität Münster, 48149 Münster, Germany Key Lab of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China § Institute of Electronics, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: All-trans retinoic acid (RA) has been proved to play important roles in regulating cell growth in various types of cells. Yet most experiments were performed by adding RA in solution previously. In this Article, we focus on the incorporation of RA, as a negatively charged moiety, into layered polyelectrolyte films on surfaces by means of layerby-layer (LbL) deposition, followed by adding of capping layers to regulate the release of RA from the films. The incorporated RA was designed to release over 5 days in buffer solution. The assembly and release of RA were verified by UV and QCM results. The controlled release of RA from multilayer films can serve as a model system to study the influence of small molecules on cell growth.

LbL deposition is a technique of fabricating polymer films through the alternating adsorption of oppositely charged polyelectrolytes. The proof of concept for LbL was first published by Gero Decher in 1991.11 Different types of methods for LbL assembly are driven by electrostatic interactions, hydrogen bonding, step-by-step reactions, sol− gel processes, molecular recognition, charge-transfer, stepwise stereocomplex assembly, and electrochemistry.12−14 However, these conventional LbL methods are not feasible for fabricating multilayers with building blocks of single charged, electrically neutral, or water-insoluble species.15,16 For the LbL assembly of RA, which is a single charged molecule in aqueous solution, unconventional LbL assembly based on electrostatic complexation was citation. The method can be described as follows: first, polyelectrolytes were mixed with single-charged molecules in aqueous solution to form electrostatic complexes; second, the complexes were deposited alternatively with counterpolyelectrolytes to form LbL films. By using unconventional LbL assembly based on electrostatic complexation, we will demonstrate how to introduce single charged molecule RA into LbL films with high loading amount and in a controllable manner. Furthermore, we will control the release of RA by changing film composition and layer structure to mimic the cell environment in vitro, which is the basis for exploring 3T3 cell growth in future.

1. INTRODUCTION Natural and synthetic small molecules have been proved to be useful chemical tools for controlling and manipulating the fates of cells by ultimately contributing to the development of effective medicines for tissue repair and regeneration. Small molecules can target signaling transduction pathways, affecting DNA replication, cell differentiation, tumor metastasis, and apoptosis.1−3 They are also important for understanding mechanistic and developmental processes. Especially, small molecules can influence 3T3 cells increasing differentiation associated with Wnt, Msx2, and TNF-α.4 Small molecules may also replace transcription factors and/or enhance the efficiency of somatic cell reprogramming.5 Study of the processes will likely provide new insights into 3T3 cells and may ultimately contribute to the development of effective medicines for adipogenesis and prevention of inflammation.6 Cell permeable small molecules such as all-trans retinoic acid (RA) have been proved to be extremely useful in modulating the fate of stem cells.7,8 RA, a small lipophilic molecule (M.W.300) derived from vitamin A, stands apart from other diffusible cell−cell signaling factors because it directs developmental processes, and it is widely used for neuronal differentiations of NIH/3T3 fibroblasts.9 Previous studies on RA modulated cell fate have been performed by adding RA in solution.10 Herein, we are interested in the effect of RA released from surface on 3T3 cells growth. For this concern, RA was loaded on PDMS surface by LbL deposition technique, to control the release of RA on a time scale accompanied by cells growth. © 2013 American Chemical Society

Received: October 26, 2012 Revised: January 15, 2013 Published: January 16, 2013 2708

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Figure 1. (a) UV−vis absorption spectra of five pair of layers of PDDA−RA/PAA multilayer films fabricated from aqueous solutions, measured after each layer was deposited. (b) Absorbance at 330 nm in UV−vis spectra lined with number of layers. (Digital Instruments), equipped with a J-scanner. Images were acquired in tapping mode at a scanning rate of 1 Hz, if not mentioned otherwise. A resonance frequency 7 kHz was chosen with low free amplitude (0.3−0.4 V) and low load upon scanning. Images were flattened when not otherwise stated. LbL Assembled RA for Cell Culture. 3T3 cells (mouse embryonic fibroblast cell line) were cultured in Dulbeccós Modified Eagle Medium (DMEM) (PAA laboratories, Pasching, Austria) supplemented with 10% fetal bovine serum (FBS, Invitrogen) in a cell culture incubator (37 °C, 5% CO2). All cells had passage numbers less than 25. At confluence, cells were detached from the culture dishes by trypsin/EDTA (0.25% trypsin/1 mM ethylenediaminotetracetic acid) solution followed by centrifugation (1200 rpm, 4 min). The pellet was resuspended in fresh medium. Before the cell seeding, the substrates were washed by phosphate buffered saline (PBS) (PAA Laboratories, Pasching, Austria). Cells were counted using a Neubauer counting chamber, and then seeded at a density of 1 × 105 cells/cm2 onto various substrates, which were placed on polystyrene tissue culture well (Falcon) in culture medium. Contact Angle Characterization. Contact angle characterization was performed using a Rame-Hart contact angle goniometer with an automated drop dispenser, image capture system, and a tilting stage. Contact angles were measured on captured images using the angle function in ImageJ. Millipore water with resistivity of >17 MΩ cm was used for all contact angle measurements. Millipore water contact angle images were captured after placing a 5 μL drop on the surface. Slip angle images were captured by tilting the stage at an angle of ∼1 o/s until the drop began to slip. The angle at whose stage was tilted was recorded as the slip angle. Images captured at the slip angle were analyzed to measure the advancing water contact angles and receding water contact angles at the leading and following edges, respectively, of the slipping drop.

2. EXPERIMENTAL SECTION Materials. RA was analytical grade and purchased from SigmaAldrich. Poly (diallyl- dimethylammonium chloride) (PDDA) (Mw ca. 100 000−200 000) and polyacrylic acid (PAA) (Mw ca. 1800) were obtained from Sigma-Aldrich and used as received without further purification. Deionized water was used for all of the experiments. Dulbecco’s Modified Eagle Medium (DMEM) was from PAA laboratories (Pasching, Austria), and fetal bovine serum was purchased from GIBCO (Carlsbad, CA). LbL Assembly and Characterization of RA Films. One mmol/ L RA (Sigma-Aldrich) was dissolved in water at pH 12 (adjusted with 1 M NaOH) and added dropwise to 1 mg/mL PDDA (Mw ca. 100 000−200 000, Sigma-Aldrich) solution in equal volume, forming PDDA−RA complexes. The microscope glass slides used in LbL assembly were cleaned by immersion in piranha solution (3:1 H2SO4/ H2O2, dangerous if contacted with organics) for 1 h, then thoroughly rinsed with deionized water prior to use. To achieve greater charge density on the surface, plasma treatment with 50 W for 20 s was applied to make the surface hydrophilic. In the assembly process, the slides were immersed in 1 mg/mL solution of PDDA−RA for 10 min, rinsed with DI water, dried under a stream of air, followed by 10 min immersion in 1 mg/mL PAA (Mw ca. 1800, Sigma-Aldrich), rinsing, and drying. The cycle could then be repeated to obtain the desired number of layers. The prepared multilayer film was denoted as (PDDA−RA/PAA)n with n referring to the number of PDDA−RA/ PAA bilayers. RA films were also deposited on PDMS films. PDMS precursors, Sylgard 184A and B (Dow Corning), were combined in a 10:1 mass ratio. Thin films were prepared by pouring the prepolymer mixture into a glass Petri dish in an evacuated vacuum until no bubbles were released and cured at 120 °C for 4 h. After plasma treatment, the PDMS surfaces were made hydrophilic by plasma treatment at 50 W for 20 s. The preparation of RA multilayer films by LbL deposition was similar to that on the glass slides. Five bilayers of PDDA−RA/PAA were assembled before more capping layers of PDDA/PAA were deposited. The LbL assembly process was monitored using an 8453 UV−vis Chem Station spectrophotometer produced by Agilent Technologies, with data collected after the deposition of each bilayer. Surface morphology of the films was obtained using a NanoScope IIIa from Bruker (Santa Barbara, CA) atomic force microscope operated in tapping mode with silicon nitride cantilevers. Quartz Crystal Microgravimetry (QCM). QCM resonators were purchased from Stanford Research Systems with electrode materials of chrome/gold and surface polished. QCM-D measurements were performed with the Q-SENSE D300 system equipped with a QAFC 301 axial flow chamber (Q-SENSE), as described in detail elsewhere. Upon interaction of soft matter with the surface of a sensor crystal, changes in the resonance frequency, f, related to attached mass, and in the dissipation, D, related to frictional losses in the adlayer were measured with a time resolution better than 1 s. All measurements were performed at a temperature of 24−25 °C. Atomic Force Microscopy (AFM). AFM measurements were performed in liquid using a Multimode Nanoscope IIIa instrument

3. RESULTS AND DISCUSSION Buildup of LbL Films Bearing RA. The PDDA−RA/PAA polyelectrolyte multilayer films were fabricated by two-step assembly that involves the formation of the PDDA−RA complex in bulk and LbL alternating deposition of PDDA− RA complex and PAA on the quartz substrate. UV−vis spectroscopy was employed to monitor the fabrication process. As shown in Figure 1a, the absorption peak at 330 nm is the Soret band of RA in PDDA−RA complex. It should be noted that the absorption peak shifts to 350 nm whenever PAA is deposited onto the PDDA−RA complex. A plausible reason is that while RA is in ionized state in PDDA−RA complex, it is recovered to its neutral state when immersed in PAA solution for the deposition of PAA layers, resulting in the shift of absorption peak. A linear increase of the absorbance at 330 nm with either the odd number or the even number of the layers is observed, which indicates a regular deposition process (Figure 1b). 2709

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Figure 2. (a) Controlled release of RA from multilayer films of (PDDA−RA/PAA)5 (PDDA/PAA)3 in 0.01 M PBS solution monitored by UV−vis spectra. Absorption spectra from top to bottom correspond to immersion time in 0.01 M PBS solution from 0 s, 5 s, 10 s, 30 s, 1 min, 10 min, 30 min, 1 h, 2 h, nd 3 h, respectively. (b) The kinetics of the release of RA. Absorbance at 330 nm was extracted form corresponding UV−vis spectra.

Figure 3. (a) UV−vis absorption spectra of 5 pair of (PDDA−RA/PAA)5 layers, measured after each deposition cycle. (b) A linear increase of the absorbance versus number of layers deposited.

Figure 4. (a) Release of RA from the (PDDA−RA/PAA)5 (PDDA/PAA)3 multilayer film in DMEM aqueous solution. (b) (PDDA−RA/PAA)5 in DMEM solution characterized by UV−vis spectra.

shown in Figures S1,2. When n = 3, it takes about 5 days for complete release, as shown in Figure 4. As compared to the release of RA in (PDDA−RA/PAA)5 without capping layers (Figure 4b), the use of capping layers dramatically slows the release process and makes it in a controlled manner. To understand more about the kinetics of the release of RA from (PDDA−RA/PAA)5 (PDDA/PAA)3 multilayer films, QCM has been used to monitor the releasing process. QCM can measure the total mass of the deposited multilayers and monitor the mass change versus immersion time of immersion in DMEM aqueous solution. As shown in Figure 5, the release of the RA is a slow and continuous process. AFM imaging has been performed to investigate the surface morphology and to study if there is any change on PDMS, with or without coatings. As shown in Figure 6a−c, the PDMS surface is flat, and the surfaces with coatings became rougher: the root-mean-square (RMS) is 0.87 nm for (PDDA/PAA)3 PDDA without loading RA, and 0.94 nm for (PDDA−RA/ PAA)5 bearing RA. However, the contact angle of these three surfaces did not show significant differences (Figure 6d−f).

To achieve the controlled release of RA, multilayer films of (PDDA−RA/PAA)5 are capped with three pair of (PDDA/ PAA)3 layers. As seen from Figure 2, the release of RA is a slow process. We have noted that the release of RA from multilayer films with capping layers can last for several hours (Figure 2b). We wondered if the above strategy of fabricating multilayer films bearing RA on quartz substrates could also work on PDMS, which is a suitable substrate for the fabrication of cell arrays because it is biocompatible, inexpensive, durable, and readily integrated with microsystems. Because of low surface energy and inherent hydrophobicity, O2 plasma treatment was applied to make PDMS changing from hydrophobic to hydrophilic, bearing negative charge on the surface for LbL assembly of PDDA−RA/PAA multilayer films. As shown in Figure 3, the absorption of RA increases with the number of layers deposited in a linear fashion, indicating that multilayer films of PDDA−RA/PAA can be fabricated on PDMS surfaces as well. The release of RA from the multilayers (PDDA−RA/PAA)5 (PDDA/PAA)n (n=1,2) on PDMS is followed by UV spectrum, as 2710

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see that 3T3 cells already successfully adhered on PDMS substrate and formed a spindle like shape, which is a normal morphology; on (PDDA/PAA)3 substrate, 3T3 cells had some trouble adhering and did not spread quite well; on (PDDA− RA/PAA)5 (PDDA/PAA)3 substrate, the adhesion of 3T3 cells is even worse. With longer time, 3T3 could spread and form flat morphology. The enormous difference in morphology is presented in Figure 6j−l after 5 days culture, while RA was completely released from substrate. Images show that 3T3 cells on PDMS were of spike spindle shape and fully confluent on PDMS, while they could only form aggregations on (PDDA/ PAA)3, but rosette clusters and produced less protrusions, as well as were less confluent on (PDDA−RA/PAA)5 (PDDA/ PAA)3 (see high magnification images on top left). Because the contact angles on three surfaces are almost the same, morphological differences of the cell should not be induced by the surface hydrophility. It has to be further studied at the cellular level to determine the meaning of the morphological variations on the three different surfaces.

Figure 5. Time courses of frequency changes ΔF observed during the release of RA from (PDDA−RA/PAA)5 (PDDA/PAA)3 multilayer films in the DMEM solution at 25 °C.

To investigate the effect of RA released from multilayer films on 3T3 cell growth, 3T3 cells were cultured in DMEM on three types of substrates for 5 days: PDMS, (PDDA/PAA)3, and (PDDA−RA/PAA)5 (PDDA/PAA)3. Cell morphology was observed during culture. Figure 6g−i showed 3T3 cells morphology in three different substrates after 24 h. We could

Figure 6. (a) AFM image of PDMS. (b) AFM image of (PDDA/PAA)3 PDDA on PDMS. (c) AFM image of (PDDA−RA/PAA)5 PDDA−RA on PDMS. (d) Contact angle of PDMS. (e) Contact angle of (PDDA/PAA)3 on PDMS. (f) Contact angle of (PDDA−RA/PAA)5 (PDDA/PAA)3 on PDMS. (g) 3T3 cells cultured on PDMS on day 1. (h) 3T3 cells cultured on (PDDA/PAA)3 on day 1. (i) 3T3 cells cultured on (PDDA−RA/PAA)5 (PDDA/PAA)3 on day 1. (j) 3T3 cells cultured on PDMS on day 5. (k) 3T3 cells cultured on (PDDA/PAA)3 on day 5. (l) 3T3 cells cultured on (PDDA−RA/PAA)5 (PDDA/PAA)3 on day 5. 2711

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(12) Zhang, X.; Chen, H.; Zhang, H. Y. Layer-by-Layer Assembly: from Conventional to Unconventional Methods. Chem. Commun. 2007, 1395−1405. (13) Schlenoff, J. B. Retrospective on the Future of Polyelectrolyte Multilayer. Langmuir 2009, 25, 14007−14010. (14) Li, Y.; Wang, X.; Sun, J. Q. Layer-by-Layer Assembly for Rapid Gerneration of Thick Polymeric Films. Chem. Soc. Rev. 2012, 41, 5998−6009. (15) Chen, H.; Zeng, G. H.; Wang, Z. Q.; Zhang, X.; Peng, M. L.; Wu, L. Z.; Tung, C. H. To Combine Precursor Assembly and Layerby-Layer Deposition for Incorporation of Single-Charged Species: Nanocontainers with Charge-selectivity and Nanoreactors. Chem. Mater. 2005, 17, 6679−6685. (16) Chen, H.; Zeng, G. H.; Wang, Z. Q.; Zhang, X. To Construct Ion Traps for Enhancing the Permselectivity and Permeability of Polyelectrolyte Multilayer Films. Macromolecules 2007, 40, 653−660.

4. CONCLUSIONS We have used unconventional LbL assembly based on electrostatic complexation to load RA into PDDA/PAA multilayer assemblies. To achieve the controlled release of RA, PDDA/PAA layers were added as capping layers on top of PDDA−RA/PAA multilayers. As a result, RA is continuously released within 5 days, which fits the cell growth time scale very well. We expect that the RA loaded PDMS substrate can be applied to direct stem cell fate in a controlled manner in the near future.

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Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the DFG-NSFC Transregio Project and EU MICROCARE project. We thank Prof. Xi Zhang at Tsinghua University for helpful discussions.

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REFERENCES

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