Enhancing Surface Coverage and Growth in Layer-by-Layer Assembly

Aug 1, 2013 - Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560012, India. •S Supporting Information. ABSTRACT: ...
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Enhancing Surface Coverage and Growth in Layer-by-Layer Assembly of Protein Nanoparticles Vaishakhi Mohanta and Satish Patil* Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560012, India S Supporting Information *

ABSTRACT: Thin films of bovine serum albumin (BSA) nanoparticles are fabricated via layer-by-layer assembly. The surface of BSA nanoparticles have two oppositely acting functional groups on the surface: amine (NH2) and carboxylate (COO−). The protonation and deprotonation of these functional groups at different pH vary the charge density on the particle surface, and entirely different growth can be observed by varying the nature of the complementary polymer and the pH of the particles. The complementary polymers used in this study are poly(dimethyldiallylammonium chloride) (PDDAC) and poly(acrylic acid) (PAA). The assembly of BSA nanoparticles based on electrostatic interaction with PDDAC suffers from the poor loading of the nanoparticles. The assembly with PAA aided by a hydrogen bonding interaction shows tremendous improvement in the growth of the assembly over PDDAC. Moreover, the pH of the BSA nanoparticles was observed to affect the loading of nanoparticles in the LbL assembly with PAA significantly.

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INTRODUCTION Layer-by-layer (LbL) assembly is a simple and versatile approach by which various functional materials (e.g., polymers, nanoparticles) can be coated onto planar as well as nonplanar surfaces.1−5 The growth mechanism of LbL-assembled thin films of polymers varying over certain parameters, mainly, ionic strength and pH, has been extensively studied by many researchers over the past decade.6−8 The ionic strength and pH remarkably affect the charge density on polymers, leading to either strengthening or weakening of the interaction between polymers and a change in the polymeric conformation. These two factors more or less decide the fate of the LbL assembly of polymers via electrostatic interaction or hydrogen bonding. However, involving a 3D particle as a component of LbL assembly requires an additional parameter to be considered, that is, the interparticle interactions.9,10 The interactions between nanoparticles and polymer/protein in dispersion media have been extensively investigated in the literature;11,12 however, the situation differs when the same is deposited on a surface because the interaction with the underlying substrate becomes important. Mainly, two forces control the deposition of nanoparticles on a substrate: the interaction of the nanoparticles with the underlying substrate, which favors higher deposition and interparticle repulsion, effectively limiting the surface coverage of the particles. The interparticle interactions in the nanoparticle dispersions are retained when the nanoparticles are deposited on the substrate by the dipping © XXXX American Chemical Society

method; as a result lateral growth becomes an important factor in addition to vertical growth in the LbL assembly of nanoparticles. Unlike the conventional LbL assembly of polymers where a deposition step leads to the formation of a uniform monolayer, complete surface coverage is never achieved for a single deposition step of nanoparticles. The multiple depositions of nanoparticles leads to the lateral expansion of the particles on the surface, the nature of which may vary depending on the type of particles, more precisely, the functional groups present on the particles. The functional groups govern the interaction among the particles as well as that with the polymer deposited in a subsequent step. Although various reports exist on incorporating inorganic nanoparticles into an LbL assembly, very few reports exist on the introduction of polymeric or hydrophilic nanoparticles that are larger in magnitude and are biologically relevant.13−16 Lyon et al. have shown the applicability of a thin film constructed from the LbL assembly of 3D microgels for the controlled pulsatile release of insulin.17 The electrostatic interaction was mainly involved in the growth of the assembly; however, other weak interactions such as hydrogen bonding and hydrophobic interaction were not considered but have been extensively employed in the LbL assembly of polymers.18,19 The surface Received: May 7, 2013 Revised: August 1, 2013

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The substrate was then washed with Milli-Q water to remove the unadsorbed polymer and then dried gently under a flow of nitrogen. The next layer was deposited by immersing the substrate in a BSA nanoparticle dispersion (0.7 mg/mL) for 6 h, followed by a washing and drying step. Subsequent layers of PDDAC and BNPs are alternately deposited by repeating the aforementioned steps with a dipping time of 1 h for each layer. Thin films of BNP/PAA were fabricated in a similar manner using PAA instead of PDDAC. The BNP/PAA assembly was fabricated at two different pH values of BNPs, pH 3.5 and pH 2, adjusted using HCl. The pH of the PAA solution is 2 in both the cases. Characterization. Quartz Crystal Microbalance. The growth of the assembly was followed by QCM. An AT-cut quartz crystal with a gold electrode coated on both sides was used. The gold surface was first modified with mercaptopropionic acid (MPA) to render a negative charge to the surface by incubating overnight in an ethanolic solution of MPA. The surface of the electrode was rinsed several times with ethanol and dried under a gentle flow of nitrogen, followed by vacuum drying to ensure the complete removal of solvent. First, a layer of positively charged polymer PDDAC was deposited by incubating in PDDAC solution (1 mg/mL), followed by rinsing with Milli-Q water and drying in a similar fashion. The subsequent layers of polymer (PAA or PDDAC) and BNPs were deposited alternately in a similar fashion. The QCM resonance frequency was measured after each deposition with an EQCM oscillator (CH Instruments). The change in the frequency of oscillation with each deposition was plotted. Scanning Electron Microscopy (SEM). Samples were dried overnight under vacuum to ensure the complete removal of moisture and were sputtered with gold prior to imaging. The images were recorded on an ULTRA 55 field-emission scanning electron microscope (Karl Zeiss). Zeta Potential Measurement. Electrophoretic mobilities were determined from a PALS Zeta Potential Analyzer, ver 3.54 (Brookhaven Instrument Corp) and were converted to zeta potentials (ζ) using Hückel’s model. All experiments were performed at 25 °C. The instrument was operated at 4.00 V with a field frequency of 2.00 Hz. The results were averaged over 3 runs, with each consisting of 30 cycles.

coverage of the nanoparticles in electrostatic-interaction-driven LbL assembly is limited because of the interparticle repulsion as a result of the high zeta potential of polymeric nanoparticles. A better growth of the LbL assembly of nanoparticles can be achieved by optimizing the growth parameters to minimize this interparticle repulsion. Thus, understanding the growth mechanism of the LbL assembly of nanoparticles can give control over the surface coverage and thickness, which affect the properties of the thin films. In this report, we have tried to tailor the growth of the LbL assembly of bovine serum albumin (BSA) nanoparticles and maximize the surface coverage and thickness of the films. BSA nanoparticles (BNPs) are biocompatible in nature and have biological importance.20 BNPs have two oppositely acting functional groups on the surface: amine (NH2) and carboxylate (COO−). The protonation and deprotonation of both functional groups control the charge density on the particle surface, and entirely different growth can be observed by varying the nature of complementary polymers and the pH of the particles. LbL-assembled thin films of BSA NPs have been fabricated using two different polymers: poly(dimethyldiallylammonium chloride) (PDDAC) and poly(acrylic acid) (PAA). The assembly formation with PDDAC is carried out at pH 8.2 for BNPs, where most of the carboxylic groups are present as carboxylate anions. The assembly is based purely on the electrostatic interaction between the carboxylate anions and ammonium groups on PDDAC. The assembly with PAA is fabricated at a much lower pH, and the interactions involved are mainly hydrogen bonding between the carboxylic groups of PAA and BSA; however, one cannot rule out the possibility of the involvement of hydrophobic interactions as well. The advantage of this assembly is that we obtain films with better surface coverage and greater thickness, which was not achieved with PDDAC. This is the first report wherein polymer/protein nanoparticles are assembled via hydrogen bonding using a layer-by-layer approach.

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RESULTS AND DISCUSSION BSA nanoparticles were synthesized by a desolvation approach.21 The scanning electron microscope (SEM) image of the drop-cast particles is provided in Figure 1. The nanoparticles have a spherical morphology and a size distribution of 115 ± 33 nm with the maximum number of nanoparticles lying in the size range of 80 to 140 nm. LbL-assembled thin films of BNPs have been fabricated by using two different polymers, poly(dimethyldiallylammonium chloride) (PDDAC) and poly(acrylic acid) (PAA). The LbL assemblies are represented as (PDDAC/BNP)n and (PAA/ BNP)n, where n denotes the number of BNP deposition steps. It should be noted that n does not correspond to the number of layers, unlike the situation in conventional LbL, because a single deposition step does not lead to the formation of a uniform monolayer in the case of nanoparticles. The solution of BNPs in Milli-Q water has a pH of 8.2. At this basic pH, most of the amine groups can be expected to be present as neutral species, NH2, whereas carboxylic groups are deprotonated, yielding negatively charged carboxylate anions (COO−). The presence of excess COO− ions is responsible for the high negative zeta potential (−44 mV) of BNPs at this pH. The negative zeta potential favors the LbL assembly of these BNPs with positively charged polymer, PDDAC. PDDAC has all hydrogens on N replaced by methyl groups (structure provided in Figure S1), and as a result, there is no possibility of hydrogen bonding and this polymer can interact only electrostatically

EXPERIMENTAL SECTION

Materials. Bovine serum albumin (BSA, fraction V) and glutaraldehyde solution (25%) were obtained from S. D. Fine Chemicals Ltd. Ethanol was obtained from Brampton (Ontario, Canada). Acetic acid was obtained from Qualigens (India). Poly(acrylic acid) (PAA) and poly(dimethyldiallylammonium chloride) (PDDAC) were procured from Sigma -Aldrich. Preparation of BSA Nanoparticles. BSA nanoparticles were prepared by a desolvation technique as reported earlier. Briefly, 8 mL of ethanol was added dropwise using a dropping funnel to a BSA solution in Milli-Q water (100 mg in 2 mL) under constant stirring at room temperature. The addition of ethanol resulted in the spontaneous formation of an opalescent suspension. Thereafter, 100 μL of 8% glutaraldehyde was added to this colloidal suspension and kept for 18 h to induce cross-linking. The resulting nanoparticles were purified by 4-fold centrifugation (12 000 rpm, 15 min) and redispersed in Milli-Q water. Layer-by-Layer Self-Assembly of BSA Nanoparticles. Thin films of BSA nanoparticles (BNPs) were fabricated via LbL selfassembly using two polymers: PDDAC and PAA. Quartz plates and glass coverslips were used as substrates for the thin film assembly. The substrates were first cleaned by treatment with piranha solution (3H2SO4:1.6H2O2) overnight. (Caution! Piranha solution should be handled with care.) It was then washed several times with Milli-Q water, followed by drying under a flow of nitrogen. The treatment with piranha also serves the purpose of inducing a more negative potential on the substrate. The first layer was deposited electrostatically by immersing the substrate in PDDAC solution (1 mg/mL) overnight. B

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Figure 3. Cross-sectional SEM image of thin film of (BNP/ PDDAC)10.

the pKa of carboxylate ions of PAA (PAA has a pKa of around 5).22 At this pH, most of the carboxylic groups are protonated as COOH, and as a result, they can form hydrogen bonds with amine and carboxylic groups present on the surface of BNPs. For assembly formation with PAA, the pH of the dispersion of BNPs was decreased to 3.5. An acidic pH is required to form the assembly because COOH of PAA will be deprotonated in the basic pH of the BNP dispersion to give anionic COO−. This may lead to the instability of the assembly from repulsion between the COO− ions of PAA and those present on the surface of BNPs at pH 8.2. At pH 3.5, it can be ensured that most of the carboxylate ions on the BNP surface are protonated as COOH. The growth of the BNP/PAA assembly was followed by a frequency change in the quartz crystal microbalance (QCM) and compared to that of the BNP/PDDAC assembly (Figure 4). The growth is tremendously enhanced when favored by the hydrogen bonding interaction between BNPs (pH 3.5) and PAA as compared to the electrostatic interaction between BNPs (pH 8.2) and PDDAC. BNPs at an acidic pH of 3.5 showed a positive zeta potential of 22.6 mV unlike the negative zeta potential in basic pH of 8.2. At acidic pH, the protonation of amine groups present on the surface of BNPs generates positive species NH3+, which results in a positive zeta potential. Although at pH 2 most of the carboxylic groups of PAA are protonated, few carboxylate anions are present, which may interact electrostatically with the NH3+ of BNPs. Thus, for BNP/PAA assembly formation one may not rule out the contribution from the electrostatic interaction along with the

Figure 1. SEM image of BSA nanoparticles drop-cast on a silicon substrate. The inset shows the histogram for the size distribution of the nanoparticles, with the maximum number of particles lying in the size range of 80 to 140 nm.

with BNPs. To ascertain the presence of positive charge on PDDAC at this pH, the zeta potential of BNPs in PDDAC solution has been measured and is strongly positive at 48.67 mV. The reversal of potential is due to the deposition of positively charged polymer on the negatively charged BNPs. The high zeta potential of both components indicates that assembly formation is favored by electrostatic interaction. It should be noted that because of the wide distribution in the size of the nanoparticles one can expect the different nanoparticles to have different values of zeta potentials, and the values mentioned in the report are statistical averages. It may be possible that because of varied zeta potentials the strength of interaction of each nanoparticle with the polymer is different. The SEM image of the surface of (BNP/PDDAC) assemblies after the 2nd and 10th deposition steps of BNPs is shown in Figure 2. It is evident that, though the lateral surface coverage is enhanced after 10 deposition steps, the vertical growth of the assembly is not very impressive. The cross-sectional SEM image of (BNP/PDDAC)10 (Figure 3) shows an average thickness of 200 nm for the film, which corresponds to approximately two bilayers of BNPs. Next, the LbL assembly of BNPs was fabricated with PAA. The solution of PAA was prepared in Milli-Q water with the pH adjusted to 2 using hydrochloric acid, which is much below

Figure 2. SEM images of the surfaces of (a) (BNP/PDDAC)2 and (b) (BNP/PDDAC)10. C

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The zeta potential of BNPs (pH 3.5) in PAA solution (pH 2) is observed to be drastically reduced to −2.43 mV. This nearzero value supports our argument that the carboxylic groups on PAA are protonated as COOH. The low charge density on PAA suggests that electrostatic interaction is insufficient to provide stability to the BNP/PAA assembly. The other interactions possible in the assembly are hydrogen bonding between the carboxylic acid groups on PAA with amine or COOH present on the surface of BNPs and the hydrophobic interaction of the PAA backbone with the hydrophobic groups of BSA. These two interactions make a major contribution to BNP/PAA assembly formation and result in better growth of the assembly. The BNPs at pH 2 in the presence of PAA show a similar zeta potential of −2.44 mV. It can be expected that the nature of interactions involved between PAA and BNPs at pH 2 is similar to that at pH 3.5. The variation in the thickness of the films at two different pH values of BNPs can be explained on the basis of interparticle interactions. The result indicates that interparticle interaction is significant in determining the growth of the LbL assembly of BNPs. The interparticle repulsion during deposition on a surface is significantly reduced at pH 3.5 because of very low zeta potential of 22.6 mV. This is also reflected in the SEM image of the surface of the film (BNP(pH 3.5)/PAA)2 provided in Figure 6a, which shows that the particles are present in agglomerations (islands). The agglomeration of BNP builds up with each deposition, giving a thicker film. A dense deposition of particles is observed after 10 depositions of BNP covering the entire substrate (Figure 6b). The zeta potential of the BSA nanoparticles at pH 2 is observed to be 37.8 mV. This higher zeta potential leads to interparticle repulsion during deposition on the surface, resulting in surface coverage that is restricted. The SEM image of the surface of the film after the second deposition of BNPs (pH 2) (Figure 6c) shows that the nanoparticles are well separated. Nonetheless, good surface coverage of particles is achieved after 10 deposition steps (Figure 6d). Thus, by employing hydrogen-bonding-directed LbL assembly with PAA as the complementary polymer, a higher loading of BSA nanoparticles is achieved.

Figure 4. Layer-by-layer assembly of BNPs with PAA and PDDAC monitored by quartz crystal microbalance.

hydrogen bonding. The possible interactions between the different functional groups on BNPs and polymers are depicted in Figure S1 (SI). Moreover, because of lower charge density on PAA at acidic pH it is expected that PAA is present in a coiled conformation23 rather than the stretched conformation of PDDAC, leading to more deposition of PAA than PDDAC as is indicated by the QCM studies. We observe a greater decrease in frequency upon the deposition of PAA compared to the deposition of PDDAC. These factors may contribute to the better growth of the BNP/PAA assembly compared to that of BNP/PDDAC. The major contribution to the growth of the assembly comes from the higher deposition of BNPs. There is a huge decrease in the QCM frequency when BNP is deposited at pH 3.5 compared to that at pH 8.2. However, when the BNP is deposited at a still lower pH of 2, the BNP deposition is less than that at pH 3.5 but still significantly higher than at pH 8.2. The QCM results were further supported by cross-sectional SEM images of the thin films shown in Figure 5. The thickness of the (BNP (pH 2)/PAA)10 film is observed to be around 800 nm compared to the 2 μm thickness of (BNP (pH 3.5)/PAA)10 assembly, a 10-fold increase in the thickness compared to that of the BNP/PDDAC assembly.

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CONCLUSIONS The various interactions in the LbL assembly of BSA nanoparticles that could aid in the better growth of the assembly have been investigated. The LbL assembly based on the electrostatic interaction with PDDAC has a poor surface

Figure 5. Cross-sectional scanning electron microscopy images showing the thickness of thin films of (a) (BNP(pH 3.5)/PAA)10 and (b) (BNP(pH 2)/PAA)10. D

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Figure 6. SEM images of surfaces of (a) (BNP(pH 3.5)/PAA)2, (b) (BNP(pH 3.5)/PAA)10, (c) (BNP(pH 2)/PAA)2, and (d) (BNP(pH 2)/ PAA)10. The insets show low-magnification images.

thank the Defense Research and Development Organization (DRDO), India, for funding through project no. ERIP/ER/ 0901110/M/1184.

coverage of particles as a result of high interparticle repulsion. The growth of the BSA nanoparticle assembly is improved when PAA is used as the complementary polymer and assembly is driven by hydrogen bonding and hydrophobic interactions; thus the role of other weak interactions in the LbL assembly of nanoparticles and polymer is emphasized. The loading of the nanoparticles is higher when the interparticle repulsion is reduced by lowering the zeta potential. Thus, the interactions of the nanoparticles with the complementary polymer as well as the interparticle interactions determine the growth of the LbL assembly of polymer/protein nanoparticles.

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(1) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232−1237. (2) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Assembly of Multicomponent Protein Films by Means of Electrostatic Layer-byLayer Adsorption. J. Am. Chem. Soc. 1995, 117, 6117−6123. (3) De Geest, B. G.; Skirtach, A. G.; De Beer, T. R. M.; Sukhorukov, G. B.; Bracke, L.; Baeyens, W. R. G.; Demeester, J.; De Smedt, S. C. Stimuli-Responsive Multilayered Hybrid Nanoparticle/Polyelectrolyte Capsules. Macromol. Rapid Commun. 2007, 28, 88−95. (4) Manna, U.; Bharani, S.; Patil, S. Layer-by-Layer Self-Assembly of Modified Hyaluronic Acid/Chitosan Based on Hydrogen Bonding. Biomacromolecules 2009, 10, 2632−2639. (5) Manna, U.; Patil, S. Borax Mediated Layer-by-Layer SelfAssembly of Neutral Poly(vinyl alcohol) and Chitosan. J. Phys. Chem. B 2009, 113, 9137−9142. (6) Ariga, K.; Hill, J. P.; Ji, Q. Layer-by-Layer Assembly As a Versatile Bottom-up Nanofabrication Technique for Exploratory Research and Realistic Application. Phys. Chem. Chem. Phys. 2007, 9, 2319−2340. (7) Li, C.; Zhang, J.; Yang, S.; Li, B.-L.; Li, Y.-Y.; Zhang, X.-Z.; Zhuo, R.-X. Controlling the Growth Behaviour of Multilayered Films via Layer-by-Layer Assembly with Multiple interactions. Phys. Chem. Chem. Phys. 2009, 11, 8835−8840. (8) Xu, L.; Ankner, J. F.; Sukhishvili, S. A. Steric Effects in Ionic Pairing and Polyelectrolyte Interdiffusion within Multilayered Films: A Neutron Reflectometry Study. Macromolecules 2011, 44, 6518−6524. (9) Ostrander, J. W.; Mamedov, A. A.; Kotov, N. A. Two Modes of Linear Layer-by-Layer Growth of Nanoparticle−Polylectrolyte Multi-

ASSOCIATED CONTENT

* Supporting Information S

Structures of polymers (PDDAC and PAA) and various possible interactions between functional groups in the LbL assembly of BNPs with PDDAC and PAA. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +91-80- 22932651. Fax: +91-80-23601310. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the support of the Centre for Nano Science and Engineering (CeNSE), Indian Institute of Science, Bangalore, for FESEM and zeta potential measurements. We E

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