Colloids and Surfaces B: Biointerfaces 121 (2014) 331–339

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Branched polymer models and the mechanism of multilayer film buildup Pradeep Waduge, Dhan B. Khadka 1 , Donald T. Haynie ∗ Nanomedicine and Nanobiotechnology Laboratory, Center for Integrated Functional Materials, Department of Physics, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA

a r t i c l e

i n f o

Article history: Received 17 February 2014 Received in revised form 15 April 2014 Accepted 4 June 2014 Available online 12 June 2014 Keywords: Buildup Dendrimer Mechanism Multilayer Peptide Thermodynamics

a b s t r a c t The “in and out diffusion” hypothesis does not provide a conclusive explanation of the buildup displayed by some polyelectrolyte multilayer film systems. Here, we report initial tests of an alternative hypothesis, on which the completion of each adsorption cycle results in an increase in the number of polymer binding sites on the film surface. Polycationic dendrimeric peptides, which can potentially bind several oppositely-charged peptides each, have been designed, synthesized and utilized in comparative film buildup experiments. Material deposited, internal film structure and film surface morphology have been studied by ultraviolet spectroscopy (UVS), circular dichroism spectroscopy (CD), quartz crystal microbalance (QCM) and atomic force microscopy (AFM). Polycations tended to contribute more to film buildup than did polyanions on quartz but not on gold. Increasing the number of branches in the dendrimeric peptides from 4 to 8 reproducibly resulted in an increase in the film growth rate on quartz but not on gold. Peptide backbones tended to adopt a ␤-strand conformation on incorporation into a film. Thicker films had a greater surface roughness than thin films. The data are consistent with film buildup models in which the average number of polymer binding sites will increase with each successive adsorption cycle in the range where exponential growth is displayed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polyelectrolyte multilayer films are of interest in fundamental research and technology development [1–4]. Applications of these films include optical coatings, coatings for cell pattering, drug delivery vehicle coatings and various other film technologies [3–9]. The use of polyelectrolytes to fabricate functional nanocomposite coatings has attracted considerable attention worldwide [1–4,10–12]. The method by which polyelectrolyte multilayer nanofilms are usually made is layer-by-layer assembly [1]. A thin film is formed by dipping a solid material with a charged surface, the substrate for assembly, into a dilute solution of oppositely-charged polyelectrolyte. Loosely bound polymer is removed from the film surface by rinsing. Additional layers are prepared in the same way, alternating the charge on the polyelectrolyte in successive adsorption steps. The result can be a highly uniform multilayer thin film of

∗ Corresponding author. Tel.: +1 813 974 7793; fax: +1 813 974 5813. E-mail address: [email protected] (D.T. Haynie). 1 Present address: Division of Science & Environmental Policy, California State University, Monterey Bay, 100 Campus Center, Seaside, CA 93955, USA. http://dx.doi.org/10.1016/j.colsurfb.2014.06.012 0927-7765/© 2014 Elsevier B.V. All rights reserved.

precisely controlled thickness, architecture and functionality. The simplest polyelectrolyte multilayer nanofilm consists of one polycation species and one polyanion. Many different polymer species are suitable for incorporation in films. A film can be fabricated on a surface of virtually any size, shape, or roughness. Nano-composites are made by substituting appropriately charged nanoparticles or other entities for polyelectrolytes [4]. There have been hundreds of experimental and theoretical studies on polyelectrolyte multilayer films by many research groups over the past two decades [1,3,5–10,12–14]. Nevertheless, significant points regarding film assembly and structure are unclear or disputed. What is known and generally accepted is that, in the typical case, the adsorbing chemical species undergo spontaneous but self-limited assembly, driven by electrostatic attraction and entropy increase under externally imposed conditions. Oppositely-charged polyelectrolytes then bind each other during the adsorption process by non-stereospecific Coulombic interactions on the film surface, releasing loosely bound counterions. The resulting entropy increase is generally greater than the entropy decrease due to the loss of translational degrees of freedom of polymers. Unknowns include details of the mechanisms of film buildup in specific cases. Explaining exponential versus linear growth in the amount of material deposited per adsorption step has

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been an aim of many studies [15–26]. Different models of buildup have been proposed. Roughly categorized as the “island” model [20,27], the “surface roughness” model [17,19] and the “in and out diffusion” model [18,21,28,29], these viewpoints are not mutually exclusive. Difficulties facing the third view have been discussed in recent work [26]. Here, we report results of a test of a film buildup hypothesis in which exponential growth involves the formation of dendritic structures on the film surface. On this view, the number of polymer binding sites increases in successive adsorption steps, because the number of incompletely charge-compensated polyelectrolyte molecules increases, giving an exponential rise in the amount of material deposited, at least in the range where such growth occurs; the growth mode could become linear at a later stage of the buildup process. Peptides were adopted as model polymers. Dendrimeric forms of poly(l-lysine) (PLL) were designed and synthesized, and multilayer films of the dendrimers were fabricated with poly(lglutamic acid) (PLGA) by electrostatic layer-by-layer assembly. The dendrimeric PLL molecules were 4- or 8-branched, each with a nominal degree of polymerization (DP) of 30. UVS and CD were utilized to monitor film buildup and polymer structure in films, QCM to measure mass deposited and AFM to characterize film surface morphology and roughness. Taken together, the data from different methods provide a consistent perspective on film buildup. 2. Materials and methods 2.1. Polymers The linear polypeptides of this study were lyophilized PLL hydrogen bromide (molecular mass 30–70 kDa, “medium”; ≤150 kDa, “large”) and lyophilized PLGA sodium salt (molecular mass 3–15 kDa, “small”; 15–50 kDa, “medium”; 50–100 kDa, “large”), all from Sigma, Inc. (USA). These polymer preparations, which are polydisperse, were utilized as received. The dendrimeric polypeptides were prepared from multiple antigenic peptide (MAP) resins for solid-phase F-moc synthesis (Calbiochem, USA) at the 25-␮mol scale. (For a discussion of MAP synthesis, see Ref. [30].)

The usual purpose of MAPs, viz. to generate polyclonal antibodies that recognize certain linear epitopes, was irrelevant here. 4-branched and 8-branched MAPs were made. About 80 mg of product was obtained in each case. MALDI–TOF mass spectrometry analysis showed mass values that represented an integral number of branches of the predicted mass. In the 8-branched synthesis product, for example, species corresponded to the predicted mass for 8 branches of DP 30, 7 branches of DP 30, 6 branches of DP 30, and so on. Qualitatively, the same result was obtained for the 4-branched synthesis product. A more detailed analysis of the distribution of dendrimeric species was not carried out; it was assumed that if the average number of branches was not greater for the 8-branched than the 4-branched species (which seems improbable), at the least the width of the distribution must be greater. The dendrimeric peptides were also characterized by dynamic light scattering (Malvern Zetasizer Nano S, United Kingdom). The results are shown in Table 1. For comparison, the contour length of a 30residue peptide is roughly 10 nm; the hydrodynamic radius will depend on pH and be largest when the probability of side chain ionization is highest.

2.2. Substrates Quartz microscope slides (50 × 25 mm2 ) from ChemGlass (USA) were cut into 25 × 10 mm2 pieces for use as substrates for film analysis by UVS, CD and AFM. Each piece was prepared for film fabrication by immersion in 1% sodium dodecyl sulfate at 80 ◦ C for 30 min, rinsing with 1% NaOH in ethanol/H2 O (50/50 v/v%) for 3 h, immersion in piranha solution (3:1 H2 SO4 and 30% H2 O2 ; considerable caution is required in the preparation and handling of this substance), rinsing with ultrapure deionized water, and drying with a stream of N2 gas. Gold-coated QCM resonators (Q-Sense, Sweden), 14 mm in diameter and less than 1 mm thick, were exposed to UV light for 10 min, immersed in 5:1:1-mixture of deionized water, 25% ammonia and 30% hydrogen peroxide at 75 ◦ C for 5 min, rinsed extensively with deionized water, and dried with a stream of N2 gas.

Table 1 Polymer species of this study. Abbreviationa

Descriptionb

4-K30

4-Branched K30

9.7 ± 0.9

8-K30

8-Branched K30

7.1 ± 0.6

PLL-M

Linear K205–479

N.D.e

PLGA-L

Linear E330–660

N.D.

PLGA-M

Linear E99–330

N.D.

PLGA-S

Linear E20–99

N.D.

a

Schematicc

Particle radius (nm)d

L = “large”, M = “medium”, S = “small”; see text for details. K = lysine and E = glutamic acid; Xn = n residues of X. c Filled circles, lysine-based MAP core. Open circles, lysine residues added by solid-phase synthesis (dendrimeric peptides) or lysine or glutamic acid residues added by solution-phase synthesis (linear peptides). d By dynamic light scattering. e N.D. = not determined. b

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2.3. Film buildup All films were prepared at ambient temperature (∼24 ◦ C) from lyophilized polymeric species following dissolution in 10 mM sodium phosphate buffer, pH 7.4. Films were built up on quartz by alternating immersion of the negatively charged substrate into 2 mg/mL polycation or polyanion for 20 min per adsorption step. For QCM, films were built up by depositing polyelectrolyte solution on AT-cut gold-coated resonators (4.95 MHz, Q-Sense, Sweden) for 20 min per adsorption step, each carried out in a 30-mm humidity chamber to control polymer concentration. The substrate surface was then rinsed three times with a large volume of buffer for 3 min each. As many as 36 deposition steps were carried out in building a single film, 18 each for the polycation and the polyanion. 2.4. Spectroscopy All UV spectra were recorded at ambient temperature with a dual-beam instrument (Jasco V-660, Japan). The scanning rate was 157 nm/min, the step size 1 nm, the spectral bandwidth 2 nm. The reference cell contained a cleaned quartz substrate in buffer. Ordinary UVS measures total apparent absorbance; the signal may include a scattering component. CD experiments were done to obtain ellipticity data on the same film samples analyzed by UVS. Spectra were recorded with an Aviv Biomedical model 215 instrument (USA). The scanning rate was 100 nm/min, the wavelength step 1 nm, the time step 1 s. 10–15 scans were collected and averaged in each case. Buffer baselines of bare quartz slides were collected with the same instrument settings as the respective film sample spectra and subtracted out. 2.5. QCM Information on multilayer film buildup was sought by QCM (Q-Sense E4 instrument). Films were built on resonators with a nominal resonator frequency of 5 MHz and rinsed between polymer adsorption steps as described above. Buffer was flowed over a resonator in the instrument at ambient temperature until the signal stabilized. The nominal fluid volume above each sensor was 40 ␮L. Resonant frequency was measured at defined points in the film fabrication process. Frequency was recorded at a sampling rate of 2.2 Hz for 5 min in the absence of flow after the signal had stabilized for at least 5 min. Data were collected simultaneously for several overtones. Frequency shift was measured as the average frequency at a given deposition step minus the average frequency of the cleaned but otherwise unmodified resonator. 2.6. AFM The same samples studied by UV and CD were dried with a gentle stream of nitrogen gas immediately prior to scanning in air at ambient temperature and humidity with a Digital Instruments Q-Scope 250 (USA) in tapping mode. The cantilever had a symmetric silicon probe tip (Tap 300-G, Bulgaria). The scan size was 10 × 10 ␮m2 , the scan rate was 1.001 Hz, and there were 256 scans per image. 3. Results 3.1. UVS In usual cases, the intrinsic chromophores in peptides that make the largest net contribution to absorbance in the 190–320 nm range are peptide bonds, which have a peak maximum near 200 nm. Representative spectra of 20-layer films made of the polymeric species indicated in Table 1 are shown in Supporting information

Fig. 1. Film buildup as monitored by UVS. All solid lines represent fits of an exponential model to experimental UVS data, A = ˛eˇN , where A is the absorbance and N is the number of adsorption steps. The values obtained for ˇ are presented in Section 4. Solid lines, dendrimeric peptide/linear peptide systems. Line colors correspond to the data in Fig. S1 in Supporting information. Broken lines, linear peptide/linear peptide systems. Squares, PLL-M/PLGA-M. Filled circles, PLL-M/PLGA-L. Triangles, PLL-L/PLGA-M. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. S1. The systems studied were 4-K30 /PLGA-L, 4-K30 /PLGAM, 4-K30 /PLGA-S, 8-K30 /PLGA-L, 8-K30 /PLGA-M and 8-K30 /PLGA-S, where in each case the polycation is given first. For instance, 4K30 /PLGA-L signifies the film system consisting of 4-branched, 30-residue PLL and “large” PLGA (see Section 2). As expected, each film system had a maximum in the absorbance spectrum near 200 nm. Films built with 8-branched PLL consistently displayed greater absorbance than the respective 4-branched PLL films in the 190–240 nm range in multiple trials involving the same peptide species. A representative spectrum of PLL-M/PLGA-L, in which both polymers were linear, is shown for comparison. Changes in photon absorption can be used to monitor film buildup and analyze its character. In the present work, film growth rate correlated with the number of branches in the cationic dendrimeric polymers and, as expected, the DP of the anionic linear polymers. Fig. 1 focuses on film absorbance at 217 nm. This wavelength approximates the location of an extremum in the corresponding CD spectra (presented below). Least-squares regression showed that the UV signal grew approximately exponentially during film buildup in each case. Fits to data points for films containing dendrimeric peptides are shown as solid lines. For comparison, buildup data for all-linear polymer systems are shown as broken lines; the values are from the same spectra as the data in Fig. 1A of Ref. [26]. Cationic dendrimers having 8 branches yielded more rapid film buildup than dendrimers having 4 branches. The relative contributions of polycations and polyanions to film buildup are compared in Fig. 2 and in Fig. S2 in Supporting information. In the case of 8-K30 /PLGA-L, for example (Fig. 2), the spectral maximum in the average optical density increment for dendritic polycation adsorption was 0.10 ± 0.05, whereas for linear polyanion adsorption the same quantity was 0.065 ± 0.015, more than 30% smaller. The trend was similar for 4-K30 /PLGA-L (0.09 ± 0.06 and 0.032 ± 0.02, respectively; Fig. 2), though the optical density increment was nearly 60% smaller for the polyanion relative to the polycation. The polycation increment was about the same for 8K30 /PLGA-L and 4-K30 /PLGA-L, but the polyanion increment was 2-fold greater for the 8-branched than the 4-branched system. The data corroborate what is already apparent from Fig. 1: the 8-K30 films grew fastest. 3.2. CD The difference in absorption of right- and left-circularly polarized light is measured by this method, and the shape of the spectrum of a peptide in the far UV can provide information on

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Fig. 2. UV absorbance difference spectra. The data are for 8-K30 /PLGA-L (top) and 4-K30 /PLGA-L (bottom). Absorbance increment on deposition of polycation (left) and polyanion (right) is shown. Solid line, average absorbance increment over 10 adsorption steps. Broken lines, plus or minus standard error from the mean absorbance increment. Note the differences in vertical scale.

the average backbone conformation. Analysis of the film spectra of present study (Fig. 3) leads to the same conclusion as before: the 8branched system grew faster than the 4-branched. The greater the UV absorbance at 217 nm, the greater the amplitude of the CD signal at the same wavelength. The shape of the CD spectra of films containing dendrimers resembles that of PLL-M/PLGA-M, also shown in Fig. 3. The spectral minimum ranged from 216 nm for 4-K30 /PLGA-S to 218 nm for 8-K30 /PLGA-M; it was 217 nm for PLL-M/PLGAM. The various spectra cross the horizontal axis near 208 nm, though at somewhat different points. The average polymer backbone conformation was essentially the same in all films, but the average conformation varied slightly with film architecture or thickness. Corresponding spectral differences are evident in the UV absorbance data (see Supporting information Fig. S2).

The signal shape throughout the far-UV range, and particularly the broad band with a minimum near 217 nm, reflects a large percentage of residues in a ␤ sheet conformation, as found in previous studies by Fourier-transform infrared spectroscopy and CD [22,23,25,28,31,32]. “Random coil” peptides in solution, by contrast, show a deep minimum near 200 nm [33]. It seems likely that the thinner films of the present study had a larger percentage of residues in an irregular conformation than did the thicker films, possibly due to a greater influence of the substrate surface on polymer behavior. 3.3. QCM Utilized to determine the amount of material deposited during or after an adsorption process, QCM is a common method of monitoring multilayer film buildup and, when certain conditions are met, measuring viscoelastic properties of deposited materials [20,27,33–36]. In the present study, the resonant frequency was measured at selected points during buildup, and the increment in resonant frequency was obtained by subtraction. Surprisingly, the cumulative frequency shift was less than 5 Hz for all six dendritic film systems; very little material became deposited at any point in the process (see Supporting information Fig. S3A). Essentially the same results were obtained on repeating the QCM buildup assay with the same peptides but independently prepared polymer solutions. A positive control experiment, by contrast, carried out with different polycations but otherwise under identical conditions, yielded clear evidence of film buildup (see Supporting information Fig. S3B). 3.4. AFM

Fig. 3. CD spectra of hydrated 20-layer films. Overlaid spectra after baseline subtraction are shown. Solid lines, dendrimeric peptide/linear peptide systems. Line colors correspond to the data in Fig. S1 in Supporting information. Dashed line, PLLM/PLGA-L. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

This approach can provide detailed information on the surface topography of a material. In the present study, height measurements were made for all six dendrimer-containing film systems following dehydration (Fig. 4). The z-scale varies from image to image to highlight surface features. Surface roughness and the apparent surface feature size tended to be greater for the 8-K30

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Fig. 4. Film surface morphology. Left-hand side, 8-K30 /PLGA samples. Right-hand side, 4-K30 /PLGA samples. Immediately to the left, control surface. In each case, the horizontal division represents 2 ␮m. The vertical scale, in nanometers, varies from sample to sample. The control was bare quartz.

than the 4-K30 films. For instance, the z-scale is twice as great for 8-K30 /L as 4-K30 /L, and the lateral dimensions of the surface features are larger. All film samples were considerably thicker and rougher than the cleaned but otherwise unmodified surface (control). Quantitative analysis of the AFM data is provided in Section 4. 4. Discussion 4.1. Polyelectrolyte multilayers These films are of widespread interest for use as coatings and nanocomposite materials in cases where both

composition and structural properties need to be defined. Applications of the technology are wide-ranging, encompassing colloid stabilization, light-emitting or photovoltaic devices, electrode modification, optical storage and magnetic films, high-density charge storage in batteries, flocculation for water treatment and paper making, functional membranes, biomaterials, coatings for control over biocompatibility, enzyme immobilization, controlled release devices, sensors and nano-reactors [1–15,37–39]. In the present work, polypeptides were studied for convenience in preparing dendrimeric species. And yet, polypeptides are studied in the present context for other reasons.

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Table 2 Summary of results of UVS, CD and AFM experiments for 20-layer films. Number of K30 branches

4

8 Control

DP of PLGA L M S L M S N/A

330–660 99–330 20–99 330–660 99–330 20–99 N/A

UV absorbance at 217 nm × 10−1

ˇ [(layers)−1 ] × 10−1

CD signal at 217 nm (mdeg)

RMS surface roughness (nm)

1.14 1.3 0.76 1.7 1.9 1.5 0

1.4 1.5 1.2 1.7 1.7 1.5 0

7.3 11.5 5.2 11 13 9.0 0

6.1 9.5 5.2 13 20 4.4 0.5

4.2. Polypeptide multilayers Multilayer films made of polypeptides are of interest for applications where chemical chirality, biocompatibility, biodegradability and specific biofunctionality are relevant, especially in biotechnology and medicine [3,13,39]. Examples include coatings for in vitro/ex vivo cell and tissue culture, coatings for medical implant devices and synthetic vaccines. The present study involved peptides not as biopolymers or functional entities, however, but model polymers. Polypeptides can be considered general polyelectrolytes to the extent that the rigidity of the peptide bond of the polymer backbone does not determine physicochemical behavior in a multilayer film context. The main purpose of the present study was to analyze the behavior of dendrimeric systems in the hope that it might provide insight into mechanisms of buildup of films made of structurally related linear polyelectrolytes. Established methods of making MAPs were convenient for producing branched structures. PLL and PLGA were selected because they are the most common polypeptides in multilayer film studies [17,18,20,25,28,32,39–43]. Both polymers are also said to diffuse in multilayer films and, according to the “in and out diffusion” hypothesis, give rise to exponential growth [18,20,21,28,29].

4.3. Data consistency The multilayer systems of this study have been analyzed by UVS, CD, QCM and AFM. All basic features of the determined behavior of these polymer systems were reproducible. Key data points from Figs. 1–4 are tabulated in Table 2. The fitted curves in Fig. 1 do not go to the origin, because A = ˛eˇN → ˛ in the limit N → 0. The only way to avoid this is to increase the number of fitting parameters. Doing this, however, does not have a significant impact on ˇ, the most important parameter of the fitting process (data not shown). Graphical comparisons of the data sets are shown in Fig. 5. It is evident that RMS surface roughness, CD signal at 217 nm and the fitting parameter ˇ from UV analysis, known as the growth exponent, showed similar trends with respect to 8-branched versus 4-branched systems.

CD analysis corroborated the view by UVS (Fig. 3 and Table 2). Specifically, CD signal amplitude at 217 nm varied linearly with UV absorbance at the same wavelength. The CD data also reveal that the scattering component of the UV signal in Fig. 1 is probably too small to be significant; the scattering magnitude is expected to be the same for both hands of photon polarization; the apparent absorbance in Fig. 1 corresponds more to the amount of material deposited and absorbance per unit thickness of material than scattering. All of the CD spectra have a roughly similar shape. This implies that the peptides in all six films had approximately the same backbone structure and that all contained a significant proportion of residues in the same conformation, ␤ sheet [25,31,33,40], consistent with interpretation of Fourier-transform infrared spectra of polypeptide multilayer films [22,31,32].

4.5. AFM An increase in film surface roughness on one side of the quartz substrate correlated with an increase in CD signal at 217 nm (essentially, the amount of material deposited). The value was greater for 8-K30 than 4-K30 films, ranging from 5.2 to 9.5 nm for the latter and 4.4 to 20 nm for the former. In Ref. [44], for comparison, the mean surface roughness of an 8-layer PLL (23.4 kDa)/PLGA (54.8 kDa) multilayer film built at pH 7.4 was 4.2 nm after dehydration. In Ref. [40], the average peak height of a 10-layer PLL (84 kDa)/PLGA (84.6 kDa) film prepared at pH 7.4 was less than 3.5 nm after dehydration. The values compare favorably with the results of the present study (Table 2).

4.4. Spectroscopy UV absorbance at 217 nm for 20-layer films had a value between 0.076 and 0.114 for the 4-K30 samples (Table 2). The corresponding values for the 8-K30 samples, which ranged between 0.15 and 0.19, were nearly 2× larger. Similarly, the exponential fitting parameter ˇ ranged from 0.15 to 0.17 for 8-K30 and 0.12 to 0.15 for 4-K30 films (Table 2); 12–20% lower for the 4-K30 films. Cationic dendrimers contributed more to film buildup than linear polyanions. The amount of polyanion added to the film per adsorption step was consistently about twice as high in the 8-K30 films (Fig. 2). From the perspective of UV absorbance at 217 nm for the 20-layer film, the fitting parameter ˇ or the amount of polyanion added per adsorption step, the 8-K30 films grew faster than 4-K30 .

Fig. 5. Graphical representations of data obtained for 20-layer films by different methods. The values plotted are from Table 2. Black dots represent experimental data points. The solid lines are provided as visual aids.

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4.6. QCM The results of these experiments illustrate limitations of an approach widely regarded as synonymous with polyelectrolyte multilayer film study. Exponential buildup of the dendrimeric film systems, which was plainly evident by UVS and CD (Figs. 1–3 and Fig. S1 in Supporting information) and consistent with the surface roughness measurements by AFM (Fig. 4), could not be detected by QCM (Supporting information). There was no way of obtaining even a crude estimate of film thickness by QCM. Apparently, quartz bound the dendrimer polycations more avidly than gold bound either polymeric species, despite cleaning (see Section 2). Film buildup in a positive control experiment involving poly(Lys, Tyr) 4:1 in place of PLL, by contrast, closely resembled that in Fig. 1 and corroborated results obtained in a previous study for PLL/PLGA and co-poly(Lys4 , Trp1 )/PLGA by UV spectroscopy [26] (see Supporting information). It appears that the peptide dendrimers, which are relatively small (Table 1), more readily formed soluble complexes with PLGA in QCM experiments than during film fabrication on quartz, resulting in little if any material remaining after rinsing. Such behavior will presumably have been due to a difference in dendrimer binding affinity to the substrate or film. Film material on both sides of the quartz slides contributed to the signal in UVS and CD analysis, whereas for QCM only one side of the resonator was available for film buildup. 4.7. Contributions to buildup The polycations of this study were dendrimers, the polyanions linear polymers. The qualitative behavior of 8-K30 and 4-K30 was the same for all three DP values of PLGA. Each dendrimer molecule occupied a relatively small and fixed volume, in solution or in a film. The radius and hydrodynamic volume of the two dendrimer species were about the same (Table 1). The number of branches differed for 8-K30 and 4-K30 , but the contour length of each branch was nominally the same. The branches will have been closer together in the 8-K30 species, and the concentration of branches increased. The driving force to form intermolecular ␤ sheets will therefore have been higher in the 8-K30 dendrimer, even if the side chains were charged. The average DP of PLGA-L, by contrast, was 495; the average contour length was ca. 190 nm. PLGA is well known to adopt a nominal random coil conformation at neutral pH and moderate ionic strength, as in the present experiment. The average radius of the statistical coil will therefore have been an order of magnitude smaller. Unlike 8-K30 and 4-K30 , the linear chains were comparatively free to adopt a huge variety of conformations, in solution or in a film, and they will have done so to maximize entropy; the driving force to form intramolecular ␤ sheets will have been comparatively low. It seems probable therefore that there was a larger loss in degrees of freedom on adsorption of a linear versus a dendrimeric species. Dendrimeric polycations in this work made a larger contribution than linear polyanions to the optical density increment, as in film systems consisting of linear PLL and linear PLGA [31]; see Fig. 2. The increment in UV absorbance due to dendrimers was about the same for 8-K30 /PLGA-L and 4-K30 /PLGA-L–after rinsing. This indicates that comparable amounts of cationic material became deposited per adsorption step in the two cases. Both species of dendrimer were deposited at the same molality, that is, the same concentration of lysine residues. The number density of dendrimers in solution was therefore about half as much for 8-K30 as 4-K30 . The driving force for adsorption of 8-K30 was lower from the point of view of particle chemical potential. Nevertheless, the 8-K30 film grew faster. The substrate or film binding affinity will have been higher for individual 8-K30 than 4-K30 molecules due to the larger

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number of ways forming favorable ionic interactions. The 2-fold greater contribution of polyanions to the buildup of 8-K30 /PLGA-L versus 4-K30 /PLGA-L (Fig. 2) is too large to be attributed to the difference in dendrimer size alone (Table 1). Nor can it be due to the DP of the polyanion, which was the same for both films. The difference in polyanion adsorption must be attributable to the odds of soluble dendrimer-linear peptide complex formation or dendrimer branching. More branches will have given a dendrimer molecule a higher binding affinity to the substrate or film and a lower probability of soluble complex formation on the subsequent linear polymer adsorption step. More branches will also have enabled bound dendrimers to bind more linear polyanion. There were twice as many branches in 8-K30 as 4-K30 , and polyanions contributed twice as much to the buildup of 8-K30 /PLGA-L as 4-K30 /PLGA-L. 4.8. Comparison with linear-peptide films It is well known that oppositely-charged polyelectrolytes having a relatively low DP will form soluble complexes more readily than polyelectrolytes with a high DP [45]. It can therefore be difficult to build up a film with low molecular weight polyelectrolytes, especially weak polyelectrolytes, for example, PLL and PLGA [26,43,46]. Ref. [26] describes films prepared from different combinations of molecular mass of PLL and PLGA. The films were not dried during the fabrication process, as in the present study. The character of buildup is consistent with the present results. More specifically, PLL-M/PLGA-M > PLL-M/PLGA-L > PLL-M/PLGAS in Ref. [26], and 4-K30 /PLGA-M > 4-K30 /PLGA-L > 4-K30 /PLGA-S and 8-K30 /PLGA-M > 8-K30 /PLGA-L > 8-K30 /PLGA-S here. Films built from PLGA-L and PLGA-M grew faster than PLGA-S (Fig. 1). Soluble complex formation will therefore have been more probable for 4-K30 /PLGA-S than the other film systems. The UV absorbance data in Ref. [46] show that the optical density of PLL/PLGA nanofilms was determined more by the DP of PLL than that of PLGA. The same seems to hold here. Fig. 1 of the present work shows 8-K30 /PLGA-L > 4-K30 /PLGA-L, and 8-K30 /PLGA-S > 4K30 /PLGA-L. That is, even though the DP was higher for PLGA-L than PLGA-S, the optical density was larger for 8-K30 /PLGA-S than 4K30 /PLGA-L. Film buildup in the present study was correlated more with dendrimeric peptide branching than the DP of PLGA. It might be guessed that the film growth rate would be larger for PLGA-L than PLGA-M for one or both cationic dendrimers. That was not the case here. Nor was it found that PLL-M/PLGA-L > PLLM/PLGA-M; instead, just the opposite [26]. It may be, then, that as the DP of a linear polyelectrolyte increases, it will be able to bind a single adsorbed dendrimer on an increasing number of branches, but in so doing the linear peptide will also tend to interact more with itself. Self-interaction will be inhibited by electrostatic repulsion between side chains and the thermodynamic unfavorability of loop formation, which constrains the polymer backbone. PLGAM may give faster growth than PLGA-L when building a film with linear PLL simply because the adsorption process will reach equilibrium more rapidly for a shorter linear polymer than a longer one. 4.9. Film buildup models Various models of film buildup have been adduced. In what may be called the “island model”, buildup occurs in two successive stages [20,27]. In the first, isolated islands are formed at disparate locations on the substrate surface. Film growth is exponential, because the film surface area will grow with successive deposition cycles proportionally to the amount of material previously deposited. In the second, the growing islands coalesce, forming a single film, the surface roughness of which could vary considerably from one region to another. In the “surface roughness model”, exponential film buildup is due to increasing film roughness [19].

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Fig. 6. Dendritic model of film buildup. (A) Schematics of 4-branched and 8-branched dendrimeric peptides. (B) The binding free energy is hypothetically the same for both dendrimeric species, so the number of binding sites is the same and the number of molecules is bound following an adsorption step is the same for both species. The number of branches available for binding linear polymer molecules in a subsequent adsorption step is greater for the 8-branched than the 4-branched species, consistent with the polycation data and the polyanion data in Fig. 2. (C) The binding free energy is hypothetically larger for the 8-branched than the 4-branched species. The number of branches available for binding is larger in the 8-branched than the 4-branched case, as before, consistent with the polyanion data but not the polycation data in Fig. 2. (D) Schematics of oppositely-charged linear peptides binding to dendrimeric peptides.

The interfacial area of the film thus increases after each deposition step, and the amount of material deposited therefore tends to increase. The “in and out diffusion” hypothesis attributes the molecular cause of exponential growth to polymer diffusion within the film. It should be clear that in this context “diffusion” is not the mere translation of polymers from the surface of the film to the interior or indeed throughout the film volume, thermal agitation providing the drive for change and resulting in a continual exchange of polyelectrolyte complexation partners, but the functioning of the film as a “reservoir” of uncomplexed polyelectrolyte molecules, which migrate to the film surface during the following polymer adsorption step [18]. On the diffusion hypothesis view, then, 4-K30 films are predicted to grow at about the same rate as 8-K30 films for a given DP of PLGA, because the size of the 4-K30 dendrimers is about the same (Table 1), giving similar diffusion constants. One could even argue that 4-K30 dendrimers, having a lower mass and fewer charged groups, would have a smaller solvent shell and therefore a higher diffusion constant. The data reported here, by contrast, show that the 8-K30 films consistently grew faster than the 4-K30 (Figs. 1–3). The “in and out diffusion” hypothesis therefore seems inconsistent with the outcome of the cases reported here (cf. Ref. [26]). In the “dendritic model” (Fig. 6), dendritic structures are hypothesized to form on the film surface, increasing the number of oppositely-charged polymers that can bind in the next polymer adsorption step. Included in the experimental support for the view is the tendency of linear polyelectrolytes to form brush-like structures on an oppositely-charged surface [47,48], and it bears some resemblance to the polyelectrolyte zone model of Ladam et al., in which tails of polymers and loops extend into solution from the film surface [49], and to the concept of an active volume of film for buildup [50]. As the DP of a polyelectrolyte increases, it will bind to a surface at an increasingly large average number of locations along the chain. If each region of an adsorbed polyelectrolyte molecule not in contact with the surface is considered a branch, then as the number of branches per polymer increases, the number of binding sites per polymer will also increase. It must be noted, however, that the tendency for kinetic effects to be relevant to film buildup surely will increase as DP increases. Polyelectrolyte binding will occur during the adsorption step, but the length of time required for the newly adsorbed polymers and the film system as a whole to reach equilibrium may be considerably longer than the duration

of the adsorption step, particularly for long polymers. Medium DP polymers may therefore give rise to faster growing films than large DP polymers, as in the present study. The dendritic model appears to agree with the experimental data presented here. Additional data will be needed, however, to assess the relative ability of the island model, the surface roughness model and the dendritic model to provide a molecular explanation of exponential film buildup. A combination of these models may provide the most complete explanation. Some polymer diffusion may occur in any of the models. The dendritic model in particular has no prohibition on the diffusion-drive translation of linear or dendritic polypeptides throughout a multilayer film under thermal agitation. What remains contentious is whether a film can act as reservoir for uncomplexed polymer and thus provide a molecular explanation of exponential buildup. Polymer diffusion may still be necessary for exponential film buildup. 5. Conclusions Polyelectrolyte nanofilms made of different dendrimeric poly(llysine) molecules and linear poly(l-glutamic acid) molecules of different DP have been studied by CD, UVS, QCM and AFM. 8branched films grew faster than 4-branched films. Buildup rate correlated with the number of branches. Twice as much linear polyanion became deposited when the dendrimeric species had 8 versus 4 branches. Increases in material deposited correlated with increases in film surface roughness. Peptides in all films tended to adopt a ␤ sheet conformation. Several film buildup models, including the “dendritic model”, agree with the experimental results. Further analysis of dendrimeric polymers may provide insight into the mechanism of film buildup for linear polyelectrolyte systems. Acknowledgments We thank the Peptide Synthesis Facility at USF for assistance with dendrimeric peptide production and analysis. The project was supported by an Established Researcher Award to DTH. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.06.012.

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