Biomaterials and Biofunctionality in Layered Macromolecular Assemblies


  • Katsuhiko Ariga,

    Corresponding author
    1. World Premier International (WPI) Research Center for Materials, Nanoarchitectonics (MANA) and Supermolecules Group, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan
    • World Premier International (WPI) Research Center for Materials, Nanoarchitectonics (MANA) and Supermolecules Group, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. Fax: +81 29 860 4832
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  • Jonathan P. Hill,

    1. World Premier International (WPI) Research Center for Materials, Nanoarchitectonics (MANA) and Supermolecules Group, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan
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  • Qingmin Ji

    1. World Premier International (WPI) Research Center for Materials, Nanoarchitectonics (MANA) and Supermolecules Group, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan
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Recent research in the field of LbL assembly is summarized and categorized as fabrication, sensing, drug release/delivery, and cell technology. Special emphasis is given to topics such as cell-membrane-mimic assembly, fabrication of free-standing biomolecular structures including protein microtubes, detection of DNA adducts and reactive metabolites, DNA hybridization analysis, sensing of toxic and bio-active chemicals, entrapment of proteins and DNA, biocomponent carriers with barcode encoding, release and delivery of DNA plasmids, multiagent delivery, smart defense capsules and oxidation-resistant films, vector introduction to cells, patterned cell culturing and microfluidic microreactors, stem cell differentiation, cellular uptake and degradation, and control of cellular apoptosis.

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Although highly sophisticated systems for information transmission and energy conversion have been developed by using advanced nanotechnologies, their efficiencies have yet to exceed those achieved by biological mechanisms. The highly evolved structures formed by functional bio-components result in energy and electron transfer with incredibly high efficiency under ambient conditions, because of the relative dispositions of those components. Thus, there is a great deal that can be learnt from biological systems for the selective mimicry of their constructions and for development of further advanced artificial functions.

Although efforts have been made to create individual biomimetic units including artificial enzymes1 and permeation-controllable membranes,2 integration of these components into more sophisticated constructions is often difficult. In particular, three-dimensional assembly of biomimetic units into the desired structures remains a challenging target. Rather, preparation of layered structures is well established and can be seen in the successful development of the Langmuir-Blodgett (LB) technique.3 This technique is not limited to archetypal lipid components so that high molecular weight species such as proteins can be immobilized into ultrathin films through the LB method.

However, the LB technique can sometimes be limited by its dependence on expensive devices. Therefore, alternative methods for immobilization of biological and biomimetic components within layered structures are required. One of the most powerful alternatives to the LB technique is the layer-by-layer (LbL) adsorption method.4 It is a simple and inexpensive method for preparation of controlled layered structures and it is applicable to a variety of materials. In this short review, recent developments in layered macromolecular assemblies containing biological components are briefly summarized.

Background of LbL Technique for Bio-Related Applications

Figure 1A illustrates an example of LbL assembly on a flat surface, where the method of electrostatic layer-by-layer assembly with a cationic polyelectrolyte and anionic particles such as protein molecules is exhibited. Adsorption of the cationic polyelectrolyte at the negatively charged surface of a solid support usually causes over-adsorption resulting in surface charge reversal under appropriate conditions. A similar process of charge reversal was proved by direct measurement of surface force.5 Subsequent adsorption of anionic particles again reverses the surface charge so that alternation of the surface charge permits continuous fabrication of the layered structure. Because this mechanism can be applied to various charged substances, there is a vast choice of available materials including biological substances such as proteins,6 nucleic acids,7 saccharides,8 and virus particles9 as well as various organic polymers,10 molecular assemblies,11 and inorganic substances.12 LbL assembly possesses the distinct advantages of simplicity and low cost. We can construct films of nanometer-scale thickness in desired sequences by using beakers and tweezers! Automatic machines for film preparation have also been proposed by Shiratori and coworkers13 and by Clark and Hammond.14

Figure 1.

(A) LbL assembly through electrostatic interaction on a flat surface. (B) LbL assembly on a colloidal particle and hollow capsule formation. Reprinted with permission from K. Ariga et al., Phys. Chem. Chem. Phys. 2007, 9, 2319. © 2007, Royal Society of Chemistry.

Many modifications of the LbL method have been developed. One of the outstanding successes can be seen in LbL assembly using colloidal particles.15 As shown in Figure 1B, LbL assembly at the surface of the colloidal core is followed by destruction of the central core resulting in hollow capsules. Template materials are not limited to colloidal particles. Thus, use of porous templates such as anodic alumina pores in LbL assembling process leads to tubular objects, and self-standing films can be obtained through LbL assembly on sacrificial (soluble) substrates.16 Spin-coating17 and spraying18 have also been combined with the conventional LbL procedure leading to facile film assembly. Interactions other than electrostatic interactions have been used for LbL assembly. For example, various LbL constructions have been achieved using metal coordination,19 hydrogen bonding,20 charge transfer,21 covalent bonding,22 and stereo-complex formation.23 Biologically important specific recognition as seen between sugar and lectin can be used for the LbL assembly,24 which allows us to construct LbL assemblies with very specific components.

Important features of the LbL assembly for biomaterials are its simplicity and mildness. The LbL assembly can be performed in aqueous medium and does not require chemically harsh conditions. LbL film structures are less densely packed than those of LB films and this is advantageous for material diffusion through the films.25 One example of practical use of the LbL films containing enzymes is shown in Figure 2, where two enzymes, glucoamylase and glucose oxidase, were assembled in the same film.26 The substrate of this “reactor” is starch, which is hydrolyzed to glucose by glucoamylase through hydrolysis of the glycoside bond. The glucose produced is converted into gluconolactone by glucose oxidase with H2O2 as a by-product. This reaction sequence could be realized because of the ease of material diffusion in the LBL films. Enhanced stability of enzymes against temperature and pH changes within the LbL films has also been demonstrated.27 Soft fixing of enzyme structures helps to suppress protein denaturation.

Figure 2.

LbL multi-enzyme reactor. Reprinted with permission from K. Ariga et al., Phys. Chem. Chem. Phys. 2007, 9, 2319. © 2007, Royal Society of Chemistry.

The main features and advantages of the LbL assembly for biomaterials were summarized above. There follows several examples of recent research including the topics of fabrication, sensing, drug release/delivery, and cell technology. Selected recently published work is presented while other excellent examples are also indicated. In order to assist the reader further, we have also cited several recent reviews.

Fabrication of Novel Bio-Composites

Because the LbL method results in flexible yet structurally resilient films, a variety of biomaterials can be integrated into custom-made layer structures. For example, lipid bilayers and related vesicle structures, which can be regarded as cell membrane mimics, have been integrated into layer-by-layer structures. Although the LbL assembly of lipid vesicles has been reported,28 lipid assemblies are rather fragile and can be destroyed through contact with polymeric materials. Therefore, lipid vesicles are not always good components for electrostatic LbL assembly. In order to overcome this difficulty, Katagiri and coworkers29 developed an organic–inorganic hybrid vesicle “cerasome” that co-possesses a ceramic-like silica surface and a lipid bilayer structure. LbL assembly between cerasome and polyelectrolytes30 and between counterionic cerasomes31 has been demonstrated with the obtained structures being regarded as mimics of multi-cellular organisms.

LbL assembly of lipid bilayer and polyelectrolytes in colloidal particles was performed by Katagiri and Caruso.32 Because the lipid bilayer can accommodate biomaterials, the combined LbL structures have potential for use in drug and gene delivery. Recently, Fournier and coworkers33 reported examples using lipid vesicles as the template for layer-by-layer assembly, which could be used as a carrier of biomolecules. In their method, biomaterials such as proteins were first encapsulated within bilayer vesicles on which polyelectrolytes were then covered. Cross-linking between two kinds of polyelectrolytes, poly(L-lysine) and poly(acrylic acid), stabilized the outer shell, and the lipid component was later extracted using detergents. Because size control of lipid vesicles is well established, size-tuned carrier capsules for biomaterials can be easily obtained by this method. The composite structure obtained prior to lipid removal can be also regarded as an interesting mimic of cell membranes. Borden et al.34 also demonstrated LbL assembly of DNA and poly(L-lysine) on lipid-coated microbubbles.

As summarized in a recent review by Caruso and coworkers,35 combination of template syntheses and LbL assembly can result in various free-standing nanostructures with layered internal structures. Li and coworkers36 demonstrated formation of free-standing microtubes of proteins, lipids, and biopolymers through LbL assembly within an anodic alumina pore template followed by template removal. For example, they prepared microtubes composed entirely of proteins. Both negatively and positively charged human serum albumin could be alternately assembled into microtubes (Figure 3). The same research team recently reported microtube formation from alginate and chitosan using a similar route.37 For these low cytotoxicity microtubes, applications in biological fields are anticipated. Atomic force microscopy (AFM) images confirmed the biodegradable nature of the assembled tubes upon immersion in pancreatin. Lu et al.38 reported recently preparation of microtubes composed of human serum albumin containing artificial heme, where binding affinities for O2 and CO were investigated. In another unique approach, Wang and Caruso39 developed the LbL technique using a mesoporous silica template. One of their efforts resulted in preparation of nanoporous structures composed of protein and polyelectrolyte.39

Figure 3.

Preparation of protein microtube through LbL assembly. Reprinted with permission from G. Lu et al., Langmuir2005, 21, 1679. © 2005, American Chemical Society.


Immobilization of bioactive enzymes within ultrathin films on electrodes is useful for preparation of biosensing systems. Rusling and coworkers40 have performed pioneering research in this field as described in their recent account. For example, they reported a DNA damage-detection system using DNA/enzyme LbL films containing myoglobin or cytochrome P450 on electrodes.41 The DNA damage was detected by square-wave voltammetry using catalytic oxidation with Ru-complex and monitoring of the binding of a Co-complex. Glucose oxidase is often used for glucose detection. Calvo et al.42 reported LbL films containing glucose oxidase and an Os-complex-appended polyelectrolyte that can operate as an electric wire to mediate electron transfer between the enzyme and the electrode. Schmidtke and coworkers43 incorporated single-walled carbon nanotubes in glucose oxidase-containing LbL films. Leblanc and coworkers44 prepared LbL films with organophosphorus hydrolase for detection and digestion of paraoxon by absorption and fluorescence measurements.

Morphologies other than thin films have been also used for biosensing. Three-dimensional nanostructures with enhanced surface areas are sometimes advantageous for bio-related reactions. Very recently, Rusling and coworkers45 proposed a unique approach for detection of DNA adducts and reactive metabolites. They prepared silica microbead bioreactors coated with DNA and enzymes such as cytochrome P450 and myoglobin through LbL assembly for measurement of reactive metabolites and DNA-adduct formation rates relevant to genotoxicity screening. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone. These enzyme-DNA-coated microbeads provided significant improvements in reactive surface area and a reduction in reaction volume over alternative methods, thus providing a superior procedure for DNA adduct detection from reactive metabolites. Knoll and coworkers46 reported design of LbL microtubes for enhanced detection of DNA hybridization. They assembled quantum-dots alternately with dendrimers in the appropriate sequence of band-gap of the quantum dots, and probe DNA fragments were attached at the surface. Hybridization of sample chromophore-labeled DNA with the probe DNA could be detected through energy-transfer processes. The detection sensitivity of the microtubes was found to be tunable by varying the assembly gradient of quantum dots. Tunable sensitivity in the LbL structures can also be expected by adjusting the distance between the quantum dots and DNA or tuning the quantum emission.

Drug Release and Delivery

Capsule structures fabricated by LbL techniques are relatively flexible. Therefore, we can anticipate their practical use as drug carriers for controlled release and delivery. In fact, various approaches have been developed.47 Lvov and coworkers48 demonstrated control of pore opening and closing of LbL microcapsules composed of polyelectrolytes by changing the surrounding solvents, resulting in encapsulation of urease. The enzymes were encapsulated based on the molecular weight-selective shell permeability, while small substrates and products of enzymatic reactions could freely penetrate the capsule wall. They developed an approach for DNA encapsulation inside a biocompatible polyelectrolyte microshell using a mixed-component core of MnCO3 and DNA/spermidine complex.49 The MnCO3 was then dissolved, resulting in biocompatible capsules containing DNA/spermidine complex. Further decomposition of the DNA/spermidine complex led to selective release of low molecular weight spermidine and complete DNA entrapment. Johnston and Caruso50 recently reported shrinkage behavior of a DNA LbL capsule. This mechanism may be useful for design of controlled release of drugs from capsule interiors.

The same research team formerly demonstrated a unique approach for extremely high enzyme loading in a nano-sized capsule. In their approach, the LBL process was conducted using enzyme crystals so that destruction of the template core resulted in a concentrated pool of the enzyme within the capsule.51 Trau and coworkers52 recently proposed entrapment of highly water-soluble materials, such as proteins, glucose, vitamin C, and inorganic salts in the solid phase, through reverse-phase layer-by-layer assembly in the organic phase. LbL assembly between neutralized polyelectrolytes in organic solution can minimize dissolution of water-soluble guest substances during the assembling process. For a more advanced analytical system, De Smedt and coworkers53 proposed use of fluorescent polystyrene microspheres encoded with “barcode” and captured antibodies (Figure 4). This system enabled them to quantify proteins in serum and plasma. Li and coworkers54 recently reported fabrication of LbL microtubes of a biodegradable polymer with magnetic nanoparticles for a DNA carrier.

Figure 4.

Barcode-attached protein detection system. Reprinted with permission from S. Derveaux et al., Anal. Chem. 2008, 80, 85. © 2008, American Chemical Society.

Volodkin et al.55 reported stable immobilization of lipid vesicles within biocompatible polelectrolytes, hyaluronic acids, and poly(L-lysine) (Figure 5). Covering phospholipid vesicle with poly(L-lysine) stabilizes the vesicle structure, resulting in successful LbL assembly with counterionic polyelectrolytes. Control of material release from these vesicles was demonstrated, where the phase transition from solid to liquid crystalline phase of the lipid bilayer upon temperature increase induced a substantial release of entrapped guests.

Figure 5.

Preparation of LbL film-containing vesicles. Reprinted with permission from D. Volodkin et al., Soft Matter2008, 4, 122. © 2008, Royal Society of Chemistry.

Controlled release functions have also been demonstrated without using capsular or vesicular objects. One of the pioneering works was reported by Lynn and coworkers.56 They realized DNA delivery and subsequent gene expression using thin film-type LbL assembly containing plasmid DNA and a synthetic degradable cationic polymer fabricated on the surfaces of planar substrates (Figure 6).56 Release of the plasmid DNA from the LbL films was transcriptionally active and promoted the expression of high levels of enhanced green fluorescent protein in the cell. In their recent research, the effect of polymer structure on drug release rate has been emphasized. Use of branched biodegradable polymers resulted in slower erosion rates.57 For example, linear poly(β-amino ester) underwent erosion in ca. 2 d, while the branched analog extended drug release time to ca. 2 weeks. They also reported long-term release of plasmid DNA over ca. 2 months using a charge-shifting cationic polymer as a component of the LbL film.58 Hammond and coworkers59 reported multi-agent delivery based on control of interlayer diffusion in hydrolytically biodegradable LbL films. Hwang, Zhuo, and coworkers60 reported tunable film degradation and sustained DNA release through reduction of the LBL films of polyelectrolytes having S[BOND]S linkage.

Figure 6.

DNA release from LbL film made of degradable polyamine. Reprinted with permission from J. Zhang et al., Langmuir2004, 20, 8015. © 2004, American Chemical Society.

Protection of entrapped guest molecules should be important when LbL films are used as drug carriers. Shutava et al.61 reported protection of the interior components of LbL films against hydrogen peroxide by inserting catalase at the outer layer of the film. Hu and coworkers62 similarly reported protection of enzymes by using a catalase-protecting layer. In pioneering work, Shutava et al.63 proposed a “smart defense microcapsule” using catalase or Fe3O4 as an antioxidant layer. Furthermore, they demonstrated antioxidant properties of the LbL films containing polyphenols or tannic acid.64

A Tool for Cell Technology

As mentioned above, the simple and moderate nature of the LbL technique makes it suitable for biological applications. The next big challenge is application of this method to cell technology and tissue engineering. Several pioneering research works have been recently proposed and have been summarized in a recent review by Kotov and coworkers.65 Several examples are also briefly introduced in this section.

Ogier and coworkers66 embedded a bioactive adenoviral vector in the LbL film composed of various polyelectrolytes such as poly(L-lysine), poly(L-glutamate), hyaluronan, poly(allylamine hydrochloride), poly(styrene sulfonate), and chitosan. The adenoviral vector preserved its transduction capability that reached nearly 95% in poly(L-lysine)/poly(L-glutamate) and poly(L-lysine)/hyaluronan LbL films. Vector uptake into receptor-deficient cells was also realized. Linderman, Takayama, and coworkers67 researched bone marrow cell culture in LbL-modified poly(dimethylsiloxane) (PDMS) microreactors. Although PDMS-based microfluidic reactors would be useful for bone marrow engineering, weak attachment of cells to the PDMS surface has been an obstacle. Modification of the PDMS surface with LbL films drastically improved cell attachment. Adherent primary bone marrow cells attached and spread best on a surface with composition of [poly(diallyldimethylammonium chloride)/clay]5 (collagen/fibronectin)2 with negatively charged fibronectin exposed on the top, and remained well spread and proliferating for at least 2 weeks. Their process could be useful for creating distinctive extracellular matrices inside PDMS bioreactors for a wide variety of cell types.

Kidambi et al.68 demonstrated patterned co-cultures of primary neurons and astrocytes on LBL films of conventional polyelectrolytes without the aid of adhesive proteins/ligands. Primary neurons attached and spread preferentially on poly(styrene sulfonate) surfaces, while primary astrocytes attached to both poly(styrene sulfonate) and poly(diallyldimethylammonium chloride) surfaces. The patterned co-culture system was used to study the neuronal response to elevated levels of free fatty acids. Elevation in reactive oxygen species levels was observed to occur earlier in the patterned co-culture system than in the separated monoculture system. Jan and Kotov69 presented an interesting possibility for LbL films in stem cell technology. They demonstrated the differentiation of environment-sensitive neural stem cells, both as neurospheres and single cells, on LbL films of carbon nanotubes and polyelectrolytes. De Smedt and coworkers70 conducted in vivo studies on cellular uptake, degradation, and biocompatibility of LbL polyelectrolyte microcapsules that were fabricated from dextran sulfate and poly(L-arginine) layers on a template of calcium carbonate microparticles. Within 16 d after subcutaneous injection, most of the microcapsules were internalized by the cells and started to be degraded. These observations indicate that the LbL microcapsules made from degradable polyelectrolytes would be suitable for drug delivery. Benkirane-Jessel and coworkers71 demonstrated control of cellular apoptosis by bone morphogenetic protein and its antagonist, noggin, which were embedded in LbL films of poly(L-glutamate) and poly(L-lysine). Their results indicate that in situ control of apoptosis during tooth differentiation mediated by these reagents embedded in a polyelectrolyte multilayer film is possible.

Techniques introduced here represent only a few of the potential possibilities for LbL assembly in cell technology. Further applications will require compositional variations and alterations in architecture for optimization of the LbL technique to specific targets. Flexibility in the combination of different biomaterials should result in various important opportunities for the treatment of tissue at different localities in the human body.

Future Perspectives

The LbL technique is compatible with biological substances because it can be conducted in an aqueous environment under mild ambient conditions. Therefore, various biomolecules including protein, DNA, and oligosaccharide-type biopolymers have been assembled in controlled layered structures by using the LbL method. The great versatility of assembly styles for this method also allows construction of varied nanostructures incorporating biomolecules. These films can be immobilized either on macroscopic flat substrates or microscopic colloidal particles.

Immobilization of biocomponents onto nanostructured supports increases its importance, leading to ultrasmall biodevices including sensors and reactors. As seen in LBL assembly within anodic alumina pores, immobilization of biomolecular LbL films within regular artificial pore supports is a relatively simple matter. Mesoporous materials with highly regular nanosized pores are also useful as supporting templates for the LbL assembly, because their pore dimensions are similar to those of biomolecules so that unusual phenomena due to confinement effects might be obtained. Although mesoporous structures have been used widely for biomolecular immobilization72 and related functions such as controlled release,73 the combination of the LbL technique and mesoporous technology74 has not been thoroughly investigated. Very recently we proposed preparation of mesoporous nanocompartment films through LbL assembly of mesoporous silica capsules (Figure 7).75 The films obtained showed stimuli-free auto-modulated release of entrapped materials through a non-equilibrium evaporation process. Fabrication of such hierarchic structures could be a key process for creation of novel concepts in bio-related functions such as drug delivery. As illustrated by the mesoporous nanocompartment films, one of the most amenable approaches for such targets is combination of the versatile and bio-compatible LbL method with nano-fabricated structures such as the mesoporous materials.

Figure 7.

Material release from mesoporous nanocompartment film. Reprinted with permission from Q. Ji et al., J. Am. Chem. Soc. 2008, 130, 2376. © 2008, American Chemical Society.


The work described in this review was partially supported by a Grant-in-Aid for Scientific Research on Priority Areas “Super-Hierarchical Structures” from MEXT, Japan, Grants-in-Aid for Scientific Research (B) from JSPS, and World Premier International Research Center Initiative (WPI Initiative) on Materials Nanoarchitectonics, MEXT, Japan.

Biographical Information

Katsuhiko Ariga is a Director of the Supermolecules Group, National Institute for Materials Science (NIMS). He received his B.Eng., M.Eng., and Ph.D. degrees from the Tokyo Institute of Technology (TIT). He was an Assistant Professor at TIT, worked as a Postdoctoral Fellow at the University of Texas at Austin, and then served as a Group Leader in the Supermolecules Project at JST. Thereafter, he worked as an Associate Professor at the Nara Institute of Science and Technology, and then entered the ERATO Nanospace Project, JST. He moved to his current position in January, 2004. His research field is based on supramolecular chemistry and surface science, including the boundary research areas of organic chemistry, physical chemistry, biochemistry, and materials chemistry. His major interests are the fabrication of novel nanostructures based on molecular recognition and self-assembly, including Langmuir-Blodgett films, layer-by-layer films, and mesoporous materials. Since October 2007, he also started working as a Principal Investigator of the World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), NIMS.

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Biographical Information

Jonathan P. Hill is a Senior Researcher in the Supermolecules Group at the National Institute for Materials Science. Prior to working in NIMS, he occupied postdoctoral positions in Tokyo (ERATO Aida Nanospace Project), University of Karlsruhe (Germany), University of East Anglia (UK), and Osaka (AIST). His research interests within supramolecular science include chemistry of the tetrapyrroles, small-molecule self-assembly in the bulk state and at interfaces, and the sensing, optical, and electronic properties of molecules.

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Biographical Information

Qingmin Ji is a Postdoctoral Researcher in the Supermolecules Group at the National Institute for Materials Science. She received her B.S. (1997) and M.S. (2001) degree in material science from NanJing University of Science and Technology, China. In 2001, she came to Japan as a Ph.D. student at the University of Tsukuba, Japan. She earned her Ph.D. (2005) in chemistry under the direction of Prof. Toshimi Shimizu. She then worked as a Postdoctoral Fellow in the Nanoarchitectonics Research Center at the National Institute of Advanced Industrial Science and Technology on the inorganic structural transcription from self-assembled lipid structures. In 2006, she moved to the National Institute for Materials Science (NIMS). Her research currently focuses on the formation of layer-by-layer films and the application of mesoporous structures as controlled-release containers.

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