Polymer Films Using LbL Self-Assembly

1.124.

Polymer Films Using LbL Self-Assembly

V Kozlovskaya and E Kharlampieva, University of Alabama at Birmingham, Birmingham, AL, USA S A Sukhishvili, Stevens Institute of Technology, Hoboken, NJ, USA ã 2011 Elsevier Ltd. All rights reserved.

1.124.1. 1.124.2. 1.124.2.1. 1.124.2.1.1. 1.124.2.1.2. 1.124.2.2. 1.124.2.2.1. 1.124.2.2.2. 1.124.2.3. 1.124.2.3.1. 1.124.2.3.2. 1.124.2.3.3. 1.124.2.3.4. 1.124.2.3.5. 1.124.2.3.6. 1.124.3. References

Introduction: Strategies for Surface Modification with Polymers Fundamentals of Multilayer Formation: LbL Thin Films Electrostatic Self-Assembly of Polymers from Aqueous Solutions Equilibrium and dynamics in PEMs: lessons from PECs Internal structure of electrostatically assembled multilayers Hydrogen-Bonded Self-Assembly of Polymers from Aqueous Solutions Assembly, internal structure, and properties of HB films Temperature-controlled HB release films LbL-Derived Hydrogels Hydrogels derived from electrostatically assembled multilayers Hydrogels derived from HB multilayers LbL-derived hydrogels via click chemistry Mechanical properties of the LbL-derived hydrogel films Applications of LbL hydrogels to controlled release of bioactive molecules Nanoparticle-containing layered hydrogel films Conclusions

Abbreviations AFM ATR-FTIR DP dPMAA EDA EDC FITC FTIR GA HA HB HPC LbL LCST Lys MC NHS PAA PAAM PAH PAMA PE PEAA

Atomic force microscopy Attenuated total reflection–Fourier transform infrared spectroscopy Degree of polymerization Deuterated poly(methacrylic acid) Ethylenediamine 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride Fluorescein isothiocyanate Fourier transform infrared spectroscopy Glutaraldehyde Hyaluronic acid Hydrogen-bonded Hydroxypropylcellulose Layer-by-layer Lower critical solution temperature Lysozyme Methylcellulose N-hydroxysulfosuccinimide Poly(acrylic acid) Poly(acrylamide) Poly(allylamine hydrochloride) Poly(dimethylaminoethyl methacrylate) Polyelectrolyte Poly(ethacrylic acid)

Symbols b dbl

Scattering length Bilayer thickness

PEC PEI PEM PEO pHcrit PHEA PLL PMAA PMAA-SH PNIPAM PSMA PSS PVA PVCL PVME PVPON Q20 QPVP SA LbL SAM TEM UCST WPEC wPEM

f+ K Q

418 419 419 419 421 422 423 424 425 425 426 427 427 427 428 428 429

Polyelectrolyte complex Poly(ethylene imine) Polyelectrolyte multilayers Poly(ethylene oxide) Critical pH value Poly(2-hydroxyethyl acrylate) Poly(L-lysine) Poly(methacrylic acid) Cysteamine-modified poly(methacrylic acid) Poly(isopropylacrylamide) Poly(styrene-alt-(maleic acid)) Poly(styrene sulfonate) Poly(vinyl alcohol) Poly(vinylcaprolactam) Poly(vinyl methyl ether) Poly(vinylpyrrolidone) 20%-quaternized poly(N-ethyl-4vinylpyridinium bromide) Quaternized poly(N-ethyl-4-vinylpyridinium bromide) Spin-assisted layer-by-layer assembly Self-assembled monolayer Transmission electron microscopy Upper critical solution temperature Water-soluble polyelectrolyte complex Weak polyelectrolyte multilayer

Fraction of positively charged units KiloDalton Wavevector

417

418

V z

Polymers

s S

Volume Distance from Si template

1.124.1. Introduction: Strategies for Surface Modification with Polymers Along with the bulk properties of biomaterials, the chemistry and structure of biomaterial surfaces are critically important to assure desired biocompatibility, wear resistance, adherence or antifouling properties. Controlled surface modification is therefore the key to developing advanced materials with rationally designed biological responses. Polymer coatings can be deposited at surfaces using traditional techniques established for depositing inorganic materials in semiconductor and microelectronics industries, which include spin-coating, physical and chemical vapor deposition, sputtering, or anodic polymerization1,2 (Scheme 1). Two other groups of techniques – chemical grafting and molecular self-assembly – include bottom–up approaches with significantly better molecularlevel control of surface architectures (Scheme 1). For example, polymer brushes of various chemistry and grafting density can be fabricated at surfaces using a chemical grafting approach which can be subdivided into growing polymer brushes via surface-initiated polymerization or grafting presynthesized polymer chains to surfaces (‘grafting from’ and ‘grafting to’ approaches, respectively).3 Another group of surface modification techniques includes molecular self-assembly which is represented by deposition of self-assembled monolayers (SAMs)4 and layer-by-layer (LbL) polymer films5,6 (Scheme 1). In the SAM approach, organic molecules are deposited at the substrate driven by the interaction between the head group of self-assembling molecules and surface-binding sites, while the choice of chemistry of molecular end-group affords great flexibility in decorating substrate surfaces with specific chemical functionalities. A similar advantage of molecular control of surface chemistry is also afforded by the chemical grafting approach. However, while both chemical grafting and SAM approaches are advantageous over more traditional polymer film deposition techniques, they are both significantly limited

Interlayer roughness Scattering density

by the requirement of specific chemistry of the substrate surface. In particular, grafting approaches require decoration of surfaces with specific functional groups or initiators capable of supporting polymerization or grafting chemical reactions. The SAM approach is even more restrictive as it involves formation of covalent Si–O or strong thiol–metal bonds between organic molecules and the surface, and usually can only be applied to a limited number of surfaces, such as silicon, aluminum oxide, or noble metals. Compared to the above surface-modification techniques, deposition of polymer films at surfaces using LbL self-assembly has emerged as a promising and versatile surface modification technique.7–12 The LbL procedure is all-aqueous based, and is not restricted by specific chemistry of substrates or depositing polymers. Indeed, conformal LbL films can be produced on substrates of virtually any shape or chemistry. Film growth occurs as polymer molecules sequentially adsorb at a surface driven by a variety of interactions in aqueous environment, for example, through electrostatic interactions, hydrogen bonding, and hydrophobic forces, or even specific ‘key-lock’ type binding between biological molecules.8,13 Recently, the versatility of the LbL technique was further extended by the demonstration that LbL films of noninteracting polymers can also be fabricated at surfaces by chemically reacting polymers using ‘click chemistry.’14 Besides earlier reported use of ‘as-is’ self-assembled multilayers, recent studies showed several promising opportunities in converting self-assembled LbL films into surface-bound functional hydrogels using postdeposition cross-linking followed by release of film components15 (Scheme 2). Such soft, functional surface coatings have a multilayer-templated, nanoscopic level of control of film thickness, and are attractive for biomedical applications such as soft hydrogel coatings, which can also serve as ‘depots’ for controlled delivery of bioactive molecules from biomaterial surfaces. In addition, LbL films can also be used as base layers for further decorations of biomaterial surfaces with polymer brushes when one needs to achieve better

Chemical grafting: polymer brushes

Traditional approaches: -chemical vapor deposition -physical vapor deposition -spin coating -anodic polymerization

Polymer films

LbL assemblies

Self-assembly Self-assembled monolayers (SAMs)

Layer-by-layer films (LbL)

Scheme 1 Various techniques for depositing polymer films at surfaces.

Scheme 2 Possible ways of functionalizing surfaces using LbL self-assembly.

Polymer Films Using LbL Self-Assembly

lubrication, biocompatibility or antifouling properties of polymer coatings. While the LbL approach has not yet made a strong appearance in everyday applications in practical biomaterials science, we believe that the technique will become a universal tool for engineering multifunctional coatings of next generation biomaterials. Therefore, in this chapter we will discuss fundamentals of depositing macromolecules at surfaces using the LbL technique, internal structure of LbL films, and postassembly modification of polyelectrolyte multilayers (PEMs) into responsive surface hydrogels, all with the central focus on understanding structure–property relationships of such coatings.

1.124.2. Fundamentals of Multilayer Formation: LbL Thin Films The fundamentals and applications of LbL films as novel types of nanoscopically structured materials are summarized in several recent reviews.13,16–19 The deposition procedure is temptingly simple and is often viewed as being applicable to virtually any pair of synthetic polyelectrolytes (PEs) or biological macromolecules such as proteins, enzymes, or nucleic acids. Indeed, a variety of interacting macromolecules have been successfully used for multilayer construction. However, here we would like to first focus on the fundamental correlation between the formation of polyelectrolyte complexes (PECs) in solutions and deposition of PEMs at surfaces, and discuss the rarely mentioned cases when such coatings cannot be constructed.

419

through formation of WPECs.32 Competitive removal of PE chains from PEM to WPEC implies that chains remain strongly associated both in the PEM and WPEC, but redistribution of chains occurs, favoring WPECs and resulting in PEM erosion. One then concludes that strong intermolecular association between a pair of PE chains is not the only prerequisite for successful film deposition. Indeed, when a PEM is brought in contact with a solution of free PE chains (a situation routinely occurring during PEM buildup), erosion of multilayers can occur. Under these conditions, one usually finds a large excess of PE in solution compared to an oppositely charged polymer within the film. For PE systems in which chain exchange occurs at the experimental time scale, such excess of charged chains in solution is most favorable for the formation of WPECs.33 Detailed understanding of competition between surface and solution as applied to PEMs requires either selective labeling of polymers and/or the application of techniques which allow chemically specific monitoring of film components, such as in situ attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy. Using this technique, effects of a number of parameters, such as the type of interacting PE chains, the ratio of their lengths, as well as ionic strength and pH of deposition solutions, can be considered to determine the likelihood of multilayer stability or erosion.22 Figure 1, top panel, shows data where 98% quaternized poly-N-ethyl-4vinylpyridinium bromide (QPVP, Mw of 330 K, a polymerization degree DPQPVP of 1600) was assembled with poly (methacrylic acid) (PMAA, Mw of 150 K, DPPMAA of 1700) at pH 8.4, when both components are strongly charged.

1.124.2.1.1. Equilibrium and dynamics in PEMs: lessons from PECs Cohen Stuart and coworkers20 have clearly emphasized the correlation between the phase behavior of PECs in solution and the deposition of PEMs. Specifically, using a PE pair of poly(dimethylaminoethyl methacrylate) (PAMA) and poly (acrylic acid) (PAA), the authors observed overshoots in the total amount of polymers within a film at the step of the polycation addition. Kovacˇevic´ et al.21 found that erosion of PAMA/PAA PEMs at ionic strength of 5 mM was explained by the formation of a negatively charged water-soluble PE complex (WPEC) or positively charged WPECs during the deposition of polyanion or polycation, respectively. Because of the interrelationships in behavior of PEMs and PECs, understanding occurrences of PEM erosion is very important as it will eventually provide materials scientists with a ‘map’ of PEM growth enabling them to rationally avoid unstable regimes during film deposition.22 Limitations of the LbL technique were recognized in cases when no interpolyelectrolyte binding occurred, such as for polymers whose charge density is lower than critical.23,24 Disruption of interpolyelectrolyte binding can also be saltinduced25 and or pH-induced.26–28 Other observations, however, were also made of removal of PE chains from the PEM when the substrate is brought in contact with PE solutions. Such chain removal has been reported by several authors29–31 and is usually related to solubilization of adsorbed chains

Amount adsorbed (mg m−2) Amount adsorbed (mg m−2)

1.124.2.1. Electrostatic Self-Assembly of Polymers from Aqueous Solutions QPVP/PMAA 40

20

0 Lysozyme/PMAA

40

20

0

0

2

4 6 Layer number

8

10

Figure 1 LbL deposition of ten layers in QPVP/PMAA (top panel) and lysozyme/PMAA systems (bottom panel). Assembly of PMAA (open symbols) and QPVP or lysozyme (filled symbols) were performed for 30 min from 0.1 mg ml
1 solution in 0.01 M phosphate buffer at pH 8. Reprinted with permission from Sukhishvili, S. A.; Kharlampieva, E.; Izumrudov, V. A. Macromolecules 2006, 39, 8873–8881. Copyright 2006 American Chemical Society.

420

Polymers

A similar failure in film deposition occurs when one tries to self-assemble a globular protein, lysozyme (Lys, Mw 14 600, pI 11.0), with PMAA at pH 8.4 where PMAA is fully ionized and Lys carries overall positive charge. As shown in Figure 1, bottom panel, Lys is completely removed from the surface to solution at the step of polyacid deposition, and adsorbed polyacid is, in turn, solubilized by Lys. In solution, strong binding between a positively charged protein and charged polycarboxylic acids was found, with formation of WPECs in excess PMAA.34 Also, solubilization of PE chains by proteins of opposite charge has been observed earlier.35,36 In contrast, enhanced deposition of polymer layers, illustrated in Figure 2 (bottom panel), is dictated by the phase diagrams of WPECs (Figure 2, top panel), which shows that formation of water-insoluble complexes is thermodynamically favorable at relatively high salt concentrations. The equilibrated amounts adsorbed depend on phase diagrams for a particular polycation/polyanion pair and may vary greatly between systems. Interestingly, while multilayer formation is prohibited in 0.01 M phosphate buffer solutions, films exhibit significant growth when the concentration of counter ions becomes very low (less than 0.003 M buffer or salt solutions). The type of PE has been shown to critically affect the binding energy and equilibration time of interacting PEs in solution. In general, polycations with high density of primary amino groups and polyanions with sulfate or sulfonate groups

QPVPs*

0.8

0.4

Amount adsorbed (mg m−2)

0.0

I

II

III

40

20

0 0.0

0.2

0.4

[NaCl] (M) Figure 2 Top panel: Fraction of QPVP remaining in supernatants (QPVP*s) of QPVP/PMAA mixtures for PMAA (Mw 350 K, DP 4000) in excess of PMAA, fþ ¼ 0.167 (circles) and in excess of QPVP (Mw 330 K, DP 1600), fþ ¼ 0.833 (triangles) plotted against concentration of added salt. Bottom panel: Total amount adsorbed of nine-layer QPVP/PMAA films deposited from solution with various salt concentrations. Concentrations of polymers were 0.04 M (in monomer units) in top panel, and 0.1 mg ml
1 in bottom panel. In all experiments, 0.01 M phosphate buffer at pH 8.4 was used. Reprinted with permission from Sukhishvili, S. A.; Kharlampieva, E.; Izumrudov, V. A. Macromolecules 2006, 39, 8873–8881. Copyright 2006 American Chemical Society.

show the strongest interpolyelectrolyte binding, resulting in inhibited chain exchange within PECs and/or PEMs. With weakly bound PE pairs – polycations containing quaternary ammonium groups and carboxylate polyanions – WPECs are easily formed, often resulting in erosion of PEMs. For easily equilibrating PEC systems consisting of polyamines with tertiary or quaternary amino groups and polyacids with carboxylate or phosphate groups, it was shown that WPECs are thermodynamically stable structures, with a measurable chain transfer rate between molecularly dispersed complexes.37 Here, we consider cases where all participating polymers are highly charged, that is, either the case of strong polyacids and polybases, or weak polyacids/polybases exposed to pH values significantly higher/lower than their pKa so that macromolecules can be considered strong PEs under these conditions. Binding of two oppositely charged PE chains results in formation of multiple polymer–polymer contacts. The multiplicity of polymer–polymer contacts within polymer complexes has major consequences for PE interactions. Therefore, the observation of chain removal during deposition of strongly charged PEs within PEMs is a common phenomenon when polycations with tertiary or quaternary amino groups and polycarboxylic acid chains are used. Recently, a quantitative approach to evaluate the binding constants K1 and Kn, where K1 is the binding constant of the interacting monomeric units, and Kn is the binding constant between two interacting chains having n polymer–polymer contacts, was proposed based on studies of competitive reactions in three-component PE solutions. Interestingly, for 3,3ionene/DNA and 3,3-ionene/PAA systems, K1 values showed only a small difference (1.012 and 1.024 for 3,3-ionene/DNA and 3,3-ionene/PAA, respectively). However, for systems with polystyrene sulfonate (PSS), much larger K1 values were found. Specifically, for PSS/poly-L-histidine systems K1 was 2, that is, twice that of DNA/poly-L-histidine38 or DNA/3,3ionene39 complexes. The large difference reflected irreversible binding of PSS with the polycation in the presence of DNA. This finding is consistent with other reports that in mixtures of polyanions, a polycation preferentially binds with macromolecules with sulfonate and/or sulfate groups such as, poly (vinylsulfonate),40 PSS,41–43 or poly(vinylsulfate).44 The presence of a small number of sulfate or sulfonate groups in the polycarboxylate polymer chains provides strong selective binding with a polycation in polyanion mixtures, even though the mixture contained polycarboxylate polyanions of much higher charge density.45,46 Strong binding of sulfate- and sulfonate-based polyanions with polycations also has a profound impact on kinetics of interpolyelectrolyte chain exchange. The first striking observation is a significant acceleration of the substitution reactions, that is, an increase in the transfer rate of polycations from carboxylate to sulfate- and sulfonate-based polyanions. The rate of chain exchange in PECs is enhanced in the presence of low molecular weight electrolytes due to screening and weakening of interpolymer electrostatic interactions.37 For example, while transfer of QPVP from QPVP/PMAA WPEC to free PMAA chains occurred at a measurable rate in 0.05 M NaCl solution, the competitive displacement of PMAA from WPEC by incoming PSS chains required one order of magnitude lower concentration of salt.42 Just as PSS is one of the strongest competitors

Absorbance at 400 nm

Polymer Films Using LbL Self-Assembly

0.8 0.6 0.4 0.2 0.0 0.0

0.2

0.4

0.6

0.8

1.0

f+ Figure 3 Turbidity of QPVP/PMAA (open circles) and PAH/PMAA (filled circles) mixtures measured as the absorbance of 0.04 M (repeat units) solutions at 400 nm as a function of the mole fraction of positively charged units fþ. The pH of 8.4 was supported by 0.01 M Trizma buffer. PMAA, QPVP and PAH with Mw of 72, 200, and 70 K, respectively, were used for the experiment. Reprinted with permission from Sukhishvili, S. A.; Kharlampieva, E.; Izumrudov, V. A. Macromolecules 2006, 39, 8873–8881. Copyright 2006 American Chemical Society.

for binding with polycations, polyamines containing primary amino groups are the strongest binders among polycations. When various polyamines were investigated for their binding with DNA, their association strength with DNA increased from quaternary to tertiary-, secondary-, and primary-amine polycations as amino groups became more sterically accessible. In addition, significant contribution of nonelectrostatic dispersive interactions to the binding energy is likely to occur in the case of PSS and/or primary-amine polycations. Figure 3 contrasts phase behavior of polycation/polyanion mixtures in solution for strongly bound and weakly bound PE pairs. In mixtures of PMAA with the primary-amine polycation, poly(allylamine hydrochloride) (PAH), insoluble complexes are formed in a wide range of polycation-to-polyanion ratios, even when the excess of PMAA or PAH units is large. In contrast, solubility behavior of PMAA mixtures with the quaternized polyamine QPVP is drastically different: precipitation occurs only when fþ is close to 0.5, that is, close to the equimolar charge ratio. Excess of PMAA or QPVP (at fþ < 0.35 and >0.6, respectively) results in WPECs solubilized by either negative or positive charge in unpaired polymer units. In contrast to QPVP/PMAA films whose deposition at a surface is greatly inhibited by exchange with solution (see Figure 1, top panel), the irreversible deposition of polymer chains at surfaces occurs under the same conditions for strongly interacting systems such as PAH/PMAA or PAH/PSS. Specifically, formation of very strong and irreversible ionic pairs affords high stability to PAH/PSS films and makes this system a favorite choice for multilayer buildup. Thus, as described above, the trends observed with multilayers directly correlate with properties of PECs in solution.

1.124.2.1.2. Internal structure of electrostatically assembled multilayers Neutron reflectometry was first utilized to study the internal structure of dip-coated strong PE LbL films containing deuterated PE marker layers.47–50 In PAH/PSS films, the intensity,

421

width, and wavevector (Q) dependence of the superlattice reflectivity peaks produced by deuterated marker layers provides a wealth of information about the quality of layering within the LbL films. Polymer cationic or anionic components with individual layer thicknesses of several nanometers were partially intermixed, but strong evidence of internal layering was also observed. For example, in PAH/PSS films assembled at high salt concentration (>0.5 M) the interlayer roughness between adjacent PE layers was found to be on the order of 1.2–1.6 nm, comprising 0.4 dbl, where dbl is the PAH/PSS bilayer thickness, indicating a well-defined layer structure with intermixing between adjacent layers of the same order as the layer thickness.49,50 Using neutron reflectometry, well-ordered layered structures were also found in the case of lipid layers51 or multilayer films made of alternating sheets of rigid cellulose crystals and flexible PAH.52 Internal layer ordering was shown to be influenced by external parameters such as pH, ionic strength, temperature, and humidity both during deposition and after postassembly treatment.6,53–56 For example, increasing ionic strength or temperature during deposition resulted in increased interfacial mixing of PAH/PSS films.57,58 Schlenoff and coworkers demonstrated that postassembly exposure of PEMs to high concentrations of salt (0.8–1.0 M NaCl) induces a significant increase in polymer interlayer mixing.59 Neutron reflectometry was also employed to resolve the structure of multilayers composed of weak PEs, which exhibit more diffuse polymer layering as compared to strong PEs.60 Asassembled films show Bragg peaks characteristic of substratemediated layering within the film, as shown in Figure 4(a), with typical ‘fussiness’ found earlier for different electrostatically self-assembled films.59,61 Neutron reflectometry applied together with in situ ATR–FTIR was used to study postassembly pH treatment of LbL films composed of weak PEs.60 When a multilayer made of PMAA and a polycation with 20% of charged units (Q20) was deposited at pH 5 and then exposed to pH 7.2, about half the PMAA amount is released, while no mass loss is observed for Q20. The mechanism of response of weak polyelectrolyte multilayers (wPEMs) to pH variation involves pH-induced accumulation of excess charge within wPEMs, when the pH changes are in the region close to the apparent pKa of a weak PE. In situ ATR–FTIR allows quantification of the pH-induced imbalance of negative to positive charges, which correlates with a rapid increase in ionization of self-assembled PMAA upon pH variations. When negative charges accumulate within the film as a result of pH variation, PMAA chains with excess charge are released into solution to bring the film charge ratio back to its original value close to unity.60 Importantly, multilayer structure of the weak PE films completely disappeared after pH-induced release of polyacid. The absence of periodic Bragg peaks in the reflectivity profile revealed mixing of the Q20 and PMAA layers, and the reduction of the total polymer layer thickness from 56 to 47 nm implies release of 35–38% of the PMAA originally present in the film (Figure 4(b)). However, after changing the solution pH back to its deposition value (pH 5) in the presence of deuterated PMAA (dPMAA), films recovered 95  5% of their original thickness, implying complete reabsorption of dPMAA (Figure 4(c)). The constant scattering-length-density profile of

Polymers

RQ4 (nm-4)

(a)

RQ4 (nm-4)

(b)

(c)

dPMAA

4 3

10-5

2 -6

10

10-7 10-4

1 (d,h)PMAA/Q20 PMAA released (pH 7.5)

0 4

SiOx

3

10-5

Si

Depleted (d,h)PMAA /Q20

2

-6

10

10-7 10-4

1 Air

0 4

PMAA reabsorbed (pH 5) (d,h)PMAA/Q20

10-5

3 2

10-6 10-7 0.0

b/V (10-4 nm-2)

As-grown (pH 5)

b/V (10-4 nm-2)

RQ4 (nm-4)

10-4

1

0.5

1.0

Q (nm-1)

1.5

0

20

40

60

b/V (10-4 nm-2)

422

0 80

z (nm)

Figure 4 Neutron reflectivity data (left) for air-dried ((PMAA/Q20)4/(dPMAA/Q20))4 films after deposition at pH 5 (a), after exposure to pH 7.5 (b) and after return to pH 5 (c) and fitted scattering-length-density profiles (right) obtained experimentally. Every fifth PMAA (Mw ¼ 22 kDa) layer is deuterated to enhance neutron contrast. Reprinted with permission from Kharlampieva, E.; Ankner, J. F.; Rubinstein, M.; Sukhishvili, S. A. Phys. Rev. Lett. 2008, 100, 128303. Copyright 2008 by the American Physical Society.

the film after reabsorption suggests a uniform distribution of this material throughout the film. Besides studies of conventional LbL films (obtained by alternating dipping from polymer solutions), neutron reflectometry was also applied to LbL multilayers constructed by spraying.62,63 It was shown that spraying does not dramatically affect layering quality. However, the sprayed layers were 30% thinner than conventional dipped LbL films obtained under the same conditions.63 Neutron reflectometry was also applied to probe the internal structure of spin-assisted layer-by-layer (SA LbL) films composed of electrostatically assembled PEs.64 SA LbL assembly has been introduced by Char and Wang as a combination of conventional LbL growth with spin coating and was shown to have several advantages over the conventional assembly.65,66 First, it enables much faster film construction as compared to dipping assembly, and thus is considered to be more ‘technologically friendly.’ Second, SA LbL films have been found to possess remarkable physical properties, such as high mechanical robustness and strength. Based on this technique, a method to obtain flexible free-standing membranes with thicknesses down to 30 nm and lateral dimensions of several centimeters was developed.67–69 Finally, SA LbL technique enables deposition of nonpolar hydrophobic moieties not possible using conventional LbL assembly.70 Application of neutron reflectometry uncovered distinct layering in SA LbL films when deposited from salt-free

solutions and showed that the degree of molecular intermixing of film components could be controlled by varying the type and concentration of salt in the deposition solutions.64 The addition of 10 mM phosphate buffer induced intermixing. The enhanced layer intermixing was explained as a result of weakening chain-to-chain interactions induced by phosphate buffer caused by an ability of phosphate ions to specifically interact with primary amino groups.21,71 Indeed, the phosphate–PAH interaction results in partial neutralization of positively charged PAH segments and the formation of a loopy structure upon chain adsorption. However, the presence of 0.1 M NaCl in the phosphate buffer restored layer stratification. In addition, conventional dipped LbL films prepared under identical conditions from buffer solution displayed a more intermixed internal structure as compared to those made by SA LbL assembly.

1.124.2.2. Hydrogen-Bonded Self-Assembly of Polymers from Aqueous Solutions Apart from LbL assembly of oppositely charged polymers, deposition of polymer films driven by the formation of hydrogen bonds has strongly emerged as a powerful technique. Hydrogen bonding is the key to defining the secondary structure and behavior of many biological molecules, including proteins and poly(nucleic acids). Synthetic chemists mimic approaches existing in nature when producing supramolecular polymeric

Polymer Films Using LbL Self-Assembly

(a)

(b)

(c)

b/V (10-4 nm-2) b/V (10-4 nm-2)

b/V (10-4 nm-2)

structures from short nonpolymeric building blocks.72,73 In the case of synthetic polymers, multiple hydrogen bonding between polymer components was demonstrated to lead to deposition of LbL films. Since the discovery of such assembly more than a decade ago,74,75 a great deal of research has been focused on the development of hydrogen-bonded (HB) films as promising surface coatings. This interest stems from the fact that HB LbL materials open new opportunities in LbL films which are harder to achieve with electrostatically assembled components. Specifically, the new attractive features of HB assembly include: (1) the possibility to include polymers which carry no charge; (2) the ease of producing films at mild pH responsive to environmental temperature and/or pH changes; (3) wide possibilities in converting HB films into single- or multiple-component ultrathin hydrogel materials. The above features enable future application of such assemblies as pH- and/or temperature-responsive drug delivery materials or release films compatible with biological tissues, as well as materials with tunable mechanical properties. In aqueous solutions, the first example of HB assembly involved polyaniline self-assembled with a number of nonionic polymers, such as poly(vinylpyrrolidone) (PVPON), poly(vinyl alchohol) (PVA), poly(acrylamide) (PAAM) and poly(ethylene oxide) (PEO)74 and a conjugated copolymer of the poly(phenylenevinylene) type containing hydroxyl groups which were capable of hydrogen bonding with amine groups of a co-self-assembled poly(ethyleneimine).76 The use of aqueous solutions for HB assembly is, however, more attractive77 as water is a friendly environment for biological and drug delivery applications. HB self-assembly of water-soluble polymers often involves a weak PE, such as a polycarboxylic acid, and a neutral polymer.78–80 The inclusion of polycarboxylic acids allows for tuning the growth of HB films via variations of film constituents and deposition conditions. Because the HB assembly involves polymers which are electrically neutral, deposition is performed at acidic pH when polyacids are fully protonated.

1.124.2.2.1. of HB films

423

Assembly, internal structure, and properties

As with other materials, HB films exhibit a close correlation between their structure and properties. Although in earlier studies HB multilayers have been assumed to be strongly intermixed,81 the first direct observation of internal layering within HB LbL was studied using neutron reflectometry.82 By applying this technique, it was shown that polymer layering deviated significantly from the ideal stratification of polymers within dense polymer layers (Figure 5(a)), and the degree of interpenetration of polymer layers, expressed as interlayer roughness, s, strongly correlates with strength of intermolecular interactions between the adjacent layers. For the dPMAA layer closest to the substrate, the value of s ranged from 35 to 60 A˚ for PVPON/PMAA, poly(N-vinylcaprolactam) (PVCL)/PMAA, and poly(N-isopropylacrylamide) (PNIPAM)/PMAA films. A new and interesting observation was that the polymer layers became more diffuse with increasing distance from the surface and as polymers deposited within the film were found in more random conformations (Figure 5(b)). For the PEO/PMAA system, films were completely interdiffused (Figure 5(c)), in agreement with weak intermolecular binding and exponential growth of PEO/ PMAA films. The key property of HB multilayers composed of neutral hydrogen-bonding polymers and polycarboxylic acids is that they can be erased by an increase in pH.80 When carboxylic groups involved in hydrogen bonding become ionized, interpolymer hydrogen bonds are disrupted, eventually causing film dissolution at a critical value of pH (pHcrit). Extreme pH variations are not required to erase these films, and film dissolution occurs at slightly acidic or neutral pH values. With weaker hydrogen-bonding systems, films dissolve at a lower pH than that for stronger bound systems. The bilayer thickness of HB films correlates with the number of interpolymer binding points, determined by the intrinsic strength of interpolymer binding. For neutral polymers of comparable length, films

dPMAA

6 SiOx

4 Si

PMAA

2 0 4

Air

PAH

PHB

2 PVPON/PMAA

0 4

d = 235 nm

2 PEO/PMAA

0

0

20

40 z (nm)

60

80

Figure 5 Hydrogen-bonded (HB) layers assuming perfect layering (a) and actual structure of HB LbL PVPON/PMAA (b) and PEO/PMAA (c) films. Scattering density S (defined as S ¼ b/V, where b is a scattering length and V is a volume) is plotted against distance from Si template (z). PAH and PHB stands for poly(allylamine hydrochloride) and hydrogen-bonding polymers (PVPON or PEO), respectively. Reprinted with permission from Kharlampieva, E.; Kozlovskaya, V.; Ankner, J. F.; Sukhishvili, S. A. Langmuir 2008, 24, 11346–11349. Copyright 2008 American Chemical Society.

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Polymers

of PMAA with PEO, poly(vinyl methyl ether) (PVME), PAAM, or poly(2-hydroxyethyl acrylate) (PHEA) show higher bilayer thicknesses than strongly bound systems, such as PMAA assembled with PVPON, PNIPAM or PVCL.77 Thicker bilayers in the case of PEO/PMAA and PVME/PMAA films are a result of more loopy conformations of self-assembled polymer chains. An analogy can be drawn with electrostatically assembled films where thicker layers are also reported for PEs with lower charge density. The morphology of HB films is also greatly affected by the type of polymer used in self-assembly. Hammond, Char and coworkers included a micelle-forming hydrophobicallymodified PEO (HM-PEO) within HM-PEO/PAA HB LbL films and demonstrated that multilayers developed grainy morphologies.83 The dependence of bilayer thickness on molecular weight is strikingly different for strongly and weakly bound polymer systems. For example, the weakly bound PEO/PAA system exhibits an approximately sevenfold increase in bilayer thickness when PEO Mw was increased from 1.5 to 20 kDa.84 1.124.2.2.1.1. Effect of polymer solution pH For both weakly bound and strongly bound HB films, bilayer thickness drastically decreased as deposition pH approached pHcrit. The effect of the deposition pH is very strong, allowing construction of HB films with bilayer thickness varying from several angstroms to hundreds of nanometers. For example, for PEO/PAA system, film thickness sharply decreased in a range of deposition pH from 2.8 to 3.5, and films could not be deposited at pH 3.5 or higher. With PEO/PMAA films, this ‘modulation window’ of film growth inhibition was shifted to slightly higher pH values.84 For stronger associated PVPON/ PAA and PVPON/PMAA polymer pairs, films could not be deposited at a pH higher than 4.085 and 4.5, respectively.77 The effect of extremely low deposition pH on PVPON/PAA film thickness has also been studied. Films deposited from solution at pH 0.2–1.0 were very rough and unusually thick, with bilayer thickness around 48 nm. These large bilayer thicknesses were explained by the collapse of PAA chains into denser and less soluble globules at the extreme pH values where PAA becomes completely uncharged.85 1.124.2.2.1.2. Ionic strength of polymer solutions There are many effects of salts on the growth of HB films. First, ions screen electrostatic charge within the film and, therefore, increase ionization of weak PEs. Secondly, they may interact with polymers in highly specific ways. Finally, the presence of salts might affect solubility of polymers. All these factors affect the individual layer thicknesses of polymers deposited within HB LbL films. In one example, the addition of monovalent and divalent salts in higher concentrations (higher than 0.5 M) during assembly of PEO/PAA systems resulted in inhibition of film growth84 probably due to increased ionization of PAA. If both polymers remain uncharged, the effects of moderate concentrations of salts on the growth of HB films include dehydration and ion–dipole interactions and are usually smaller than those in electrostatically assembled systems. For example, in the case of strongly associated PVPON/PMAA films, film thickness increased by only 10% when self-assembly was performed in 0.5 M NaCl solution.77 Finally, salt can dramatically decrease the solubility of hydrophobic polymers resulting

in thicker layers within the film, as in the case of deposition of a relatively hydrophobic poly(styrene-alt-(maleic acid))86 within PEO/PSMA films, which gave layers twice as thick in 0.2 M NaCl as compared to those in 0.02 M NaCl solutions. 1.124.2.2.1.3. Temperature of polymer solutions Caruso and coworkers demonstrated that PNIPAM/PAA films deposited from PNIPAM solutions86 at 30  C were of higher thickness and much lower roughness compared to films prepared at 10 or 21  C.86 Larger amounts of PNIPAM were deposited within the films when the lower critical solution temperature (LCST) of PNIPAM (32  C) was approached and PNIPAM became less soluble in water. The behavior of other temperature-responsive polymers, PVME (LCST 36  C87) and PVCL (LCST 35  C),88 was similar.77 Bilayer film thickness increased with temperature even for films composed of PEO or PVPON and PAA77 whose LCST is significantly higher than the temperature of deposition solutions (e.g., LCST of PEO is 100–150  C depending on molecular weight).89 Such an increase is due to a positive entropy of binding PEO with polycarboxylic acids,90 which leads to a stabilization of HB multilayers at high temperatures. However, if HB films contain a polymer pair with upper critical solution temperature (UCST), thinner layers are deposited within LbL films at higher temperatures.91 One example is the PAAM/PAA system, for which the UCST of 20–25  C was reported in the case of PAAM/PAA interpenetrated networks.92 As temperature is raised above UCST, intermolecular hydrogen bonds between the polyacid and PAAM dissociate, resulting in thinner polymer layers within HB LbL films. 1.124.2.2.1.4. Concentration of polymer solutions In addition to all the parameters listed above, thickness of HB films is also affected by the concentration of assembly solutions.93 Similar to electrostatically assembled films, deposition of HB PVPON/PAA films at higher concentrations of assembly solutions resulted in larger amounts of polymer adsorbed at each deposition cycle.93

1.124.2.2.2.

Temperature-controlled HB release films

Along with pH responsiveness, temperature can be used as an important trigger in manipulating properties of HB films containing temperature-responsive components. Application of HB PNIPAM/PMAA films as temperature-triggered platforms for releasing biological materials from surfaces has been explored.94 Specifically, cells with attached patches (or ‘backpacks’) of electrostatically assembled LbL films were harvested from the substrate as a result of lowering the solution temperature to selectively dissolve the PNIPAM/PMAA surface stack and to release cellular backpacks from surfaces. Generality and the mechanism of such temperature-triggered release were studied.95 Specifically, it was shown that deposition temperature of HB films containing PNIPAM, PVCL, or PVME as a temperature-sensitive neutral component plays a crucial role in pH-triggered dissolution of the films. For example, the critical pH of (PNIPAM/PMAA)5 film disintegration shifted from 5.5 to 5.8 and to 6.1 when films were constructed at 10, 23, and 30  C, respectively. Such drastic effect of assembly temperature on pH-stability of multilayer HB films of temperature-responsive polymers indicates stronger binding

Polymer Films Using LbL Self-Assembly

of PNIPAM segments with PMAA at higher temperatures due to increased hydrophobic interactions.82 This also correlated with the almost doubled bilayer thickness of PNIPAM/PMAA films deposited at 30  C compared to that at 10  C (8.7 nm vs. 4.4 nm, respectively). Importantly, temperature of postassembly solutions also critically affects the pH-stability of HB LbL films. For example, when PNIPAM/PMAA films built at pH 2 and 23  C were immersed in solutions with increasingly high pH values at 10, 23, and 37  C, the stability of the films went up from pH 5.2 to 5.8 and to 5.9, respectively. The increased film stability at temperatures close to the LCST is correlated with decreased solubility of PNIPAM and also with the hydrophobic stabilization of hydrogen bonding between PNIPAM and PAA. When poly(carboxylic acid)s were assembled with PAAM, a polymer with a UCST, the opposite effect of temperature on multilayer stability was observed. For example, the value of pHcrit decreased when temperature was raised from 10 to 37  C due to the increased solubility of PAAM at a higher temperature which resulted in weakening intermolecular hydrogen bonds within the film. No such temperaturetriggered change in the film pH stability was observed for HB films which did not contain temperature-responsive polymers. Importantly, by selecting different pairs of hydrogen-bonding polymers from a pool of neutral polymers and polycarboxylic acids, the working pH range for such temperature-triggered release films can be controlled and adjusted to neutral and slightly basic pH values. Specifically, the use of poly(ethacrylic acid) (PEAA) instead of PAA or PMAA in film assembly enabled construction of HB LbL films which can be released by applying temperature as a trigger at a near-physiologic pH. This feature makes this approach promising for future biomedical and tissue engineering applications.

1.124.2.3. LbL-Derived Hydrogels Hydrogel matrix provides an ideal environment for hosting a variety of functional molecules such as drugs or proteins. Unlike macroscopic slab hydrogels, thin hydrogels (<100 nm) enable fast response to external stimuli.96–98 At the same time, in contrast to polymer brushes,99–101 SAMs or monolayers of adsorbed polymers, surface hydrogels possess high loading capacity to functional compounds. Such a combination of desirable properties is particularly valuable for applications in biotechnology. Electrostatic interactions between polymer layers in a multilayer assembly are often used in fabrication of ionic LbLderived ultrathin hydrogel films and membranes. Although highly swollen hydrogel-like structures can be fabricated using postassembly pH variations of non-cross-linked wPEM,91 such films often lose their structure and rearrange after long-term exposure to extreme pH environments. HB multilayers, on the other hand, often readily dissolve at neutral and slightly basic pH values. Therefore, cross-linking is usually applied to convert LbL films to surface hydrogels. The LbL-type surface hydrogels can be fabricated using sequential chemical cross-linking during self-assembly,102,103 co-self-assembly of linear polymers with microgel particles,104,105 or postassembly treatment of preconstructed LbL films via thermal-, photo-, or chemical cross-linking.106,107 Conventionally assembled electrostatic or HB LbL films are

425

converted into ultrathin hydrogels via cross-linking between self-assembled polymer chains of the same type, or between the two different types of polymers. The use of weak PEs for LbL hydrogel fabrication enables the use of pH to control loading and to trigger release of functional molecules from such matrices.

1.124.2.3.1. Hydrogels derived from electrostatically assembled multilayers Glutaraldehyde (GA) is often used as a cross-linking agent in fabricating LbL-derived ultrathin hydrogels films because of its high solubility in water.108 Cationic single-component LbL hydrogel films and capsules were initially synthesized by using selective cross-linking of chitosan by GA within chitosan/PAA multilayers.109 The GA-treated chitosan/PAA films were exposed to carbonate buffer at pH 9 overnight to induce the pH-dependent swelling of the chitosan/PAA membranes as ionization of the polyacid increased. Such a treatment resulted in complete PAA removal from the cross-linked films leaving behind chitosan hydrogel LbL-derived films or hollow capsules. Swelling of the capsules was observed when the pH was changed from the neutral to acidic with a transition at pH 5.5. By simply changing the cross-linking time, a different degree of cross-linking was achieved. A similar strategy was applied to various multilayer systems of electrostatically interacted weak PEs. For example, GA was also used to selectively cross-link PAH within PAH/PSS capsules via formation of a Schiff base between the aldehyde and the amine groups in PAH.110 After cross-linking and exposure to pH ¼ 12, the capsules slowly released PSS molecules from the cross-linked networks. After a 2-h cross-linking with GA and partial release of PSS, the permeation coefficient of these films to dextran molecules of Mw 250 kDa decreased threefold as compared to the initial PAH/PSS capsules, probably due to partial polymerization of GA. In another work, a similar strategy was applied to selectively cross-link branched poly(ethylene imine) (PEI) within PEI/PAA LbL capsule membrane.93 The resultant crosslinked PEI capsule walls contained physically trapped PAA chains. Two-component LbL hydrogels can also be fabricated if both film constituents contain reactive functional groups. Carbodiimide chemistry is generally exploited for covalent cross-linking between amine or hydroxyl and carboxylic group-containing moieties of the PE components. For instance, the chemical cross-linking of hyaluronic acid/PAH (HA/PAH) multilayer films and capsules with a mixture of water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (NHS) produced biocompatible and noncytotoxic LbL-derived HA–PAH assemblies stable under low and high pH conditions.111 If non-cross-linked, these HA–PAH assemblies dissolved due to ionization-induced swelling at pH <2 or >7.2. Biocompatible chitosan–alginate LbL capsules assembled from natural polysaccharides on calcium carbonate particles were also chemically stabilized with the GA treatment.112 Both components, chitosan and alginate, were present in the capsule wall after GA cross-linking as confirmed by the FTIR. Along with the GA cross-linking, carbodiimide chemistry was applied to directly cross-link neighboring layers of chitosan and alginate to increase stability of the films toward

Polymers

1.124.2.3.2.

Hydrogels derived from HB multilayers

HB LbL assembly of neutral polymers and poly(carboxylic acids) is another route of fabrication for ultrathin hydrogels. HB multilayers are uniquely suited for constructing responsive hydrogel films since polymer–polymer hydrogen bonds can be easily dissociated by exposure to mild values of pH. In contrast, in electrostatically assembled films, dissociation of ionic pairs is more problematic and, if possible, requires exposure to extreme values of solution pH. Therefore, stabilization of HB multilayers through covalent bonds has been recently developing as a novel technique in building ultrathin LbL hydrogel materials with unique properties.115 The approach extended the use of HB films to neutral and basic range of pH, making such materials promising candidates for biomedical or biosensing applications. Several cross-linking strategies have been explored to produce stable hydrogels from HB films, including chemical,116 thermal,107 or photo-cross-linking.107 Carbodiimide chemistry has been widely applied to selectively cross-link poly(carboxylic acids) with ethylenediamine (EDA) within polyacid/ nonionic polymer HB films or capsules.117–119 The use of nonfunctionalized PVPON, PVCL, or PEO in the HB LbL assembly resulted in complete release of nonionic polymer from the cross-linked films upon exposure to high pH, yielding single-component polyacid hydrogel films or capsules. Using this route, single-component PAA, PMAA, or PEAA hydrogel membranes were derived from the corresponding HB LbL assemblies. An astounding feature of these membranes is that in a wide range of pH values there is no extensive noncovalent binding between units of poly(carboxylic acid), and that capsule walls, or the surface-attached LbL films exist as highly hydrated (up to 300% swellable at pH 7.5) ultrathin hydrogels. Also, cross-links were introduced between both components within HB multilayer films and capsules. Rubner and coworkers have reported on PAAM/PAA or PAAM/PMAA hydrogel-like films and capsules fabricated via thermal, photo- or carbodiimide-facilitated formation of amide links between polymer counterparts.120–122 Hydrogels with strongly enhanced swelling at high pH values were also produced using PVPON- and PVCL-based copolymers. To that end, PVPON- or PVCL-based copolymers with a fraction of units containing amino groups (PVPON-co-NH2 or PVCL-co-NH2, respectively)123 were assembled with poly(carboxylic acids) through hydrogen bonding. Following exposure to solutions of water-soluble carbodiimide, amide links formed between

PVPON-co-NH2 (or PVCL-co-NH2) and polyacid chains, resulting in two-component hydrogel-like films and capsules.119,123 Significant differences were found between single- and two-component hydrogel capsules derived from HB assemblies.119,123 In the case of two-component capsules, when the pH was decreased below the critical value, hydrogen bonds between a neutral counterpart and poly(carboxylic acid) units were reformed. In the case of a single-component hydrogel system, lowering the pH-induced deswelling of such hydrogel capsule walls as a consequence of decreased polyacid ionization. Remarkably, both types of capsules reversibly changed their size in response to variations in pH and/or ionic strength (Figure 6). At the same time, distinct swelling hysteresis occurred in the case of two-component capsules (inset in Figure 6). In particular, larger capsule sizes were detected when the pH was lowered compared to those when the pH was increased. Significantly, at basic pH values, both types of hydrogel capsule walls did not contain HB polymers and were highly swollen. Recently, hydrogen bonding between PAA and methylcellulose (MC) has also been successfully used as a platform for preparation of thin hydrogel films of 600 nm thickness.124 Advantageously, these films could be cross-linked by heating at 120  C for 2–6 h, and swelled at high pH producing freely floating films. HB capsules of hydroxypropylcellulose/PAA (HPC/PAA)125 dissolved at pH higher than 3.3, and were converted into the pH-stable capsules with hydrogel-like walls through carbodiimide cross-linking between hydroxyl groups of HPC and carboxylic groups in PAA. These two-component LbL-derived hydrogels continuously swelled in the pH range

1

2

C 6 2 5 6.0 Capsule diameter (mm)

enzymatic erosion and to reduce the release rate of encapsulated indomethacin.113 Apart from chemical cross-linking, thermal treatment of electrostatically assembled LbL films and capsules has been applied as a way to stabilize LbL systems against pH-induced dissolution.114 One example is thermal cross-linking of CaCO3deposited (PAH/PAA)4 microcapsules at 180  C for 2 h.114 The thermal treatment stabilized the polymer network through the formation of PAH–PAA amide linkages. Interestingly, the capsules kept their shape even when severely swollen in highly acidic solutions. Under these low pH conditions, electrostatic pairing between PAH and PAA was disrupted, and the capsule walls were converted to positively charged ultrathin hydrogels capable of binding polyanions, such as PSS.114

Capsule diameter (mm)

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pH Figure 6 Confocal laser scanning microscopy (CLSM) images of (PVPON-co-NH2-20/PMAA)7 (1) and (PMAA)7 (2) capsules at pH 2, scale bar is 1 mm; pH dependence of the diameter of (PVPON-NH2-20/ PMAA)7 (curve 1, filled circles) and (PMAA)7 capsules (curve 2, open squares) cross-linked for 18 h. The inset shows hysteresis of (PVPON-NH2-20/PMAA)7 capsule size upon increasing (filled circles) and decreasing (open triangles) pH. Reprinted with permission from Kozlovskaya, V.; Sukhishvili, S. A. Macromolecules 2006, 39, 5569–5572. Copyright 2006 American Chemical Society.

Polymer Films Using LbL Self-Assembly

from 3 to 5 due to deprotonation of the carboxylic groups. The HPC/PAA cross-linked hydrogels required 5–15 days to reach the swelling equilibrium. The prolonged swelling kinetics observed for both lightly and heavily cross-linked HPC/PAA capsules was due to the hydrophobic nature and significant rigidity of the HPC chains.

LbL-derived hydrogels via click chemistry

An elegant approach to prepare single-component ultrathin LbL films and capsules of PAA via click chemistry was introduced by Caruso and coworkers.14,126 In click chemistry, covalent reactions with high yields can be performed under mild conditions. Using azide- or alkyne-modified PAA polymers, stepwise growth of LbL films was demonstrated in the presence of copper(I) and sodium ascorbate in aqueous solution.14 The deposition was promoted by the formation of 1,2,3-triazole cross-links.126 However, the requirement for the use of copper in the azide–alkyne click chemistry reaction prevents the use of this approach for biological applications. Thiol–ene reaction was also explored for the fabrication of LbL hydrogels.127 First, thiol-PMAA and ene-PMAA were LbL-assembled with PVPON and the PMAA layers were then cross-linked via thiol–ene reaction initiated by UV-light. The release of PVPON from the film at pH 7 resulted in layered PMAA hydrogel films.

1.124.2.3.4. Mechanical properties of the LbL-derived hydrogel films Mechanical properties of pH-responsive hydrogel-like capsules were investigated using atomic force microscopy (AFM) single capsule force spectroscopy measurements.128 The microcapsule stiffness of one- and two-component hydrogel capsules was dramatically pH-dependent. In the case of cross-linked PVPON-co-NH2/PMAA capsules at pH 2, intermolecular hydrogen-bonding interactions of the hydrogel network dominated, and the capsule walls were rigid. Dramatic softening of (PVPON-co-NH2/PMAA)7 capsules occurred in a narrow range of pH between 5.4 and 6, as intermolecular hydrogen bonds dissociated at higher pH values (Figure 7). The corresponding decrease in stiffness of the walls was from 18  9 to 1.4  0.5 mN m
1 for pH 5.4 and 6, respectively. A decrease in stiffness from 550  150 to less than 1 mN m
1 also occurred for single-component (PMAA)10 hydrogel capsules. The large difference between the systems was due to the different capsule wall thickness of 20 and 7 nm for (PMAA)10 and (PVPON-co-NH2/PMAA)7, respectively. The important finding was made that mechanical properties of LbL-derived hydrogels can be designed as switchable in response to environmental stimuli, with the switch triggered at mild pH conditions. This possibility of switching mechanical properties holds a significant potential for designing future-generation controlled delivery membranes and containers. In a different system, a nanoindentation approach employing colloidal probe AFM was used to study the effect of crosslinking on the elastic properties of cross-linked poly(L-lysine)/ hyaluronan (PLL/HA) films.129 Higher concentrations of carbodiimide resulted in a larger number of cross-links within the film and enhanced film stiffness. Similar modulation of stiffness was used to tune cell adhesion properties of cross-linked films of a poly(carboxylic acid) and PAAM.130 Highly

700 650 600 550 500 kshaI (mN m–1)

1.124.2.3.3.

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Figure 7 Reversibility of the softening process for cross-linked (PVPON-co-NH2-20/PMAA)7 (open circles) and cross-linked (PMAA)10 (filled circles) microcapsules for pH changes from 5.4 to 6 or 6.5, respectively. Reproduced from Elsner, N.; Kozlovskaya, V.; Sukhishvili, S. A.; Fery, A. Soft Matter 2006, 2, 971, with permission from The Royal Society of Chemistry.

swellable, with a swelling degree of more than 300%, low Young’s modulus cross-linked films of PAAM/PAA and PAAM/PMAA were resistant to cell adhesion.

1.124.2.3.5. Applications of LbL hydrogels to controlled release of bioactive molecules Hydrogels derived from HB LbL films are promising materials capable of pH-controlled loading and release of functional compounds. Specifically, loading of proteins and heparin within LbL-derived surface-attached PMAA hydrogels was studied in situ using ATR–FTIR and ellipsometry.15 The loading of Lys, a protein with an isoelectric point of 11.5, within the PMAA hydrogels was fast and saturated after 15 min when 0.1 mg ml
1 protein solutions were used. Almost 98% of Lys loaded at pH 7.5 released when the gel was exposed to pH 4 and carboxylic groups became completely protonated. Excellent correlation of the amount of Lys loaded within the film with the charge density of the PMAA hydrogels points to an essentially electrostatic nature of protein interactions with the hydrogel matrix. A similar trend of reversible binding of positively charged protein within PMAA hydrogels was obtained with another protein, ribonuclease. The hydrogel mesh size was large enough to allow transport of Lys globules through the hydrogel matrix. Negatively charged macromolecules such as heparin could also be included within the amphoteric hydrogels. The amount of heparin loaded at pH 3 was proportional to PMAA hydrogel thickness. Figure 8 contrasts the inclusion of heparin and Lys within surface hydrogels at various pH values. As in the case of Lys, heparin adsorption was irreversible towards dilution with buffer solutions at a constant pH. However, heparin was effectively released from the PMAA hydrogel upon an increase in pH, when the hydrogel carboxylic groups deprotonated and acquired negative charge.

428

Polymers

Heparin

40 4 20 2

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pH Figure 8 pH dependence of amounts of Lys (open triangles) and heparin (filled triangles) absorbed within (PMAA)5 hydrogel as inferred from in situ ATR–FTIR and confirmed with ellipsometry. pH values were supported by 0.01 M phosphate buffer solutions. Reprinted with permission from Kharlampieva, E.; Erel-Unal, I.; Sukhishvili, S. A. Langmuir 2007, 23, 175–181. Copyright 2007 American Chemical Society.

In a similar way, at physiologic pH values of 7.5, the most abundant plasma protein, albumin, could not be included into the PMAA matrix due to size and charge considerations. A possibility of constructing functional containers with biodegradable hydrogel walls has also been explored. The walls of such containers consisted of single-component PMAA hydrogel cross-linked via biodegradable disulfide links,131 produced by treatment of HB multilayers of PVPON and cysteaminemodified PMAA (PMAA-SH) with hydrogen peroxide.132 The PMAA capsules stabilized with disulfide bonds were able to load proteins, oligonucleotides, or DNA and then to release their cargo under reducing conditions.133,134 For example, encapsulated fluorescently labeled protein, FITC-transferrin was released after deconstruction of the capsules in the presence of thiol–disulfide exchange reagent – dithiothreitol.

1.124.2.3.6. films

Nanoparticle-containing layered hydrogel

The LbL-derived hydrogel matrices in their swollen states possess large number of functional groups able to attract various metal or metal-containing ions. This advantage can be successfully used for the creation of nanoparticle-loaded thin hydrogel coatings by incorporating presynthesized nanoparticles within such hydrogels, or by using the hydrogels as templates for in situ synthesis of nanoparticles. In situ synthesis of nanoparticles has been applied to carbodiimide-cross-linked PAAM/PAA HB precursor matrices to fabricate silver-loaded ultrathin hydrogel films and capsules.120,121 Metal ions were bound to the carboxylic acid groups when the films were suspended in aqueous solutions of silver acetate and Ag was reduced in the presence of borohydride. Multiple ion loading and reduction cycles were performed to increase nanoparticle size. While the above approach of using carboxylic groups of the hydrogel as binding sites for metal precursor ions is useful for the synthesis of Ag/hydrogel composites, it cannot be used for in situ synthesis of gold nanoparticles, as hydrogel

2 mm Figure 9 Top: In situ synthesis of gold nanoparticles within PMAA hydrogel capsules derived from PVPON/PMAA hydrogen-bonded capsules through cross-linking with ethylenediamine. Bottom: Transmission electron microscopy (TEM) Images of cross-linked hydrogel capsules after gold reduction within the shell. Reprinted with permission from Kozlovskaya, V.; Kharlampieva, E.; Chang, S.; Muhlbauer, R.; Tsukruk, V. V. Chem. Mater. 2009, 21, 2158–2167. Copyright 2009 American Chemical Society.

carboxylate groups and [AuCl4]
ions are both anionic. The direct synthesis of gold nanoparticles within layered PMAA hydrogel capsules was demonstrated for hydrogels cross-linked with EDA.135 The [AuCl4]
ions were bound with a fraction of amino groups of one-end-tethered EDA cross-linker within the hydrogel. Well-dispersed gold nanoparticles of 10  2 nm were obtained within PMAA hydrogels (Figure 9). Importantly, the pH-responsive properties of the PMAA hydrogel capsules were preserved after the formation of gold nanoparticles. Combining unique properties of metal nanoparticles (localized surface plasmons, thiol-based tethering of biomolecules to nanoparticle surfaces, or antibacterial properties in case of silver nanoparticles, enhancement of mechanical rigidity, biocompatibility and nontoxicity of gold nanoparticles) with responsive behavior of ultrathin LbL-derived hydrogels can result in materials crucially important in biosensing and delivery of functional cargo.

1.124.3.

Conclusions

Recent developments in LbL-based surface modifications demonstrate the power of this technique in engineering surface

Polymer Films Using LbL Self-Assembly

coatings with controlled thickness, architecture, nanoscopic structure, and mechanical properties. The technique is applicable to the deposition of a broad range of functional polymers at a variety of surfaces including biomaterials, and the deposition procedures for LbL coatings may be varied from sequential dipping to spin-coating66 or spraying.136,137 The use of chemical approaches to LbL films, such as postassembly cross-linking, or chemical reactions during polymer assembly, enables fabrication of soft hydrogel surface coatings with controlled thickness and swelling capabilities. Such highly swollen surface coatings are excellent candidates for controlling cellular adhesion and spreading, or endowing surfaces with antifouling properties. Furthermore, LbL hydrogels can be used as versatile matrices for loading and controlled release of bioactive molecules. Versatility of the LbL approach and its value to modification of biomaterial surfaces can be further enhanced by combining the LbL technique with chemical grafting of polymer chains, including polypeptide sequences, to the LbL matrix. Such surface constructs will be capable of dual control of biomaterial interaction with biological tissues by delivering bioactive compounds from the bulk of the film, while additionally controlling cellular adhesion through grafted chemical moieties. These opportunities make LbL films promising candidates for modification of biomaterial surfaces.

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