Multilayer microcapsules with tailored structures for bio-related applications

Abstract

The technique for fabrication of hollow multilayer microcapsules is initially established by layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes onto sacrificial colloidal particles followed by core removal. It has the advantages of precise control over the size, shape, composition, wall thickness and functions of the obtained hollow microcapsules. More recently, the technique has been developed fast from the fabrication and basic property investigation to functionalization and applications, in particular in bio-related fields such as controlled drug delivery, biosensors and bioreactors. In this article, we shall review recent advances in property manipulation of electrostatic and hydrogen bonded microcapsules by chemical crosslinking, and microcapsules assembled by other driving forces such as covalent interaction, base pair interaction, host–guest interaction and van der Waals interaction. Then the possible applications of LbL microcapsules in bio-related fields are summarized with particular emphasis on very recent achievements in terms of loading and release, surface modification for stealth function or targeting and use as biosensors and bioreactors.

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Weijun Tong

Weijun Tong is currently a lecturer of material science at Zhejiang University. He obtained his PhD in material science in 2007 under the supervision of Prof. Changyou Gao at Zhejiang University, China and Prof. Helmuth Möhwald at the Max-Planck Institute of Colloids and Interfaces, Germany. His main scientific interests are supramolecular assembly and microcapsules and their applications in the biomedical field.

Changyou Gao

Changyou Gao is currently a professor of material science at Zhejiang University. He got his PhD in polymer chemistry and physics in 1996 under the supervision of Prof. Jiacong Shen at Jilin University, China. His research interests include self-assembled microcapsules, biomaterials and nanomaterials for biomedical applications.

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1. Introduction

Hollow capsules are of great interest due to their potential applications and fundamental importance as new colloidal structures in areas such as medicine, catalysis, cosmetics as well as biotechnology.¹ Recently, a novel type of hollow microcapsule has been fabricated by means of layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes^2,3 on colloidal templates, followed by core removal (Fig. 1).^4–6 Using this technique, capsules with well-controlled size and shape, finely tuned wall thickness and variable wall compositions have been produced.⁷ Moreover, microcapsules with customized physicochemical properties can be obtained by incorporation of one or more functional species such as biomacromolecules,⁸ lipids,⁹ photoactive dyes,¹⁰nanoparticles,¹¹ as well as multivalent ions¹² onto the capsule walls or into the capsule interiors. The LbL microcapsules with integrated multifunctionalities have a high capacity for loading a wide range of substances and a sensitive response to diverse stimuli, thus they are highly attractive for bio-related applications.^13–15

Fig. 1 Schematic illustration of the polyelectrolyte deposition process and of subsequent core decomposition. Reprinted with permission from ref. 5. Copyright 1998, Wiley-VCH.

Up to now, several excellent articles have reviewed the progress of LbL microcapsules with respect to fabrication and physicochemical properties,^7,16 permeability,^17–19 loading and release,^18–21 serving as microreactors,^19,22 mechanical properties^23–25 and biofunctionality.^26,27 In particular, the inorganic reactions inside the polyelectrolyte microcapsules to synthesize magnetic, fluorescent and mineral nanoparticles have clear relevance to the bio-related applications, which has been covered by a recent comprehensive review.²² Readers can get the respective overviews there and those works shall not be repeated here. In this article, we focus on the very recent progress of the LbL microcapsules with respect to manipulation of their properties by chemical crosslinking, fabrication of the microcapsules based on new driving forces such as covalent interaction, base pair interaction, host–guest interaction and van der Waals interaction, and their bio-related functions and applications for loading and release, anti-endocytosis or targeting, biosensors and bioreactors.

2 Advances in fabrication of multilayer microcapsules by LbL techniques

2.1 Crosslinking to manipulate the properties of LbL microcapsules based on electrostatic interaction and hydrogen bonding

Although electrostatic interaction is generally strong enough to hold the integrity of LbL microcapsules, it is still susceptible to high ionic strength, extreme pH and strong polar organic solvents, and the properties of microcapsules may thus become adverse, consequently influencing the applications. Crosslinking of the capsule walls is an effective way to manipulate their properties such as permeability, mechanical properties and stability. Several crosslinking methods have been developed, most of which were initially used for multilayer films and subsequently for LbL microcapsules. For example, Bruening and co-workers used a simple heat induced amide formation method to crosslink the poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayers.²⁸ The crosslinked, nylon-like films are stable over a wide pH range and highly impermeable. Zhang and co-workers constructed diazoresins (DARs) and poly(sodium styrene sulfonate) (PSS) multilayers, which can form a covalently crosslinked structure by UV irradiation.²⁹ The films have no detectable damage when sonicated in H₂O–dimethyl formamide (DMF)–ZnCl₂ ternary solvent for 0.5 h, while the uncrosslinked films lose 30% of their material when incubated in the same solvent for 5 min. These methods are also applicable to microcapsules and can induce stable structures. For example, the PAH/PAA microcapsules crosslinked by amide formation at high temperature are rather stable in a wide pH range.³⁰ The DAR/PSS microcapsules crosslinked by UV radiation have a higher mechanical stability measured by a method of osmotic-induced invagination,^31,32 and are more stable in various chemical environments than their uncrosslinked counterparts.³³ The crosslinked DAR/PSS capsules can also effectively reduce the cut-off molecular weight of the capsule walls, thus enzymes can be encapsulated after the crosslinking.³⁴ Moreover, carbodiimide chemistry has also been used to crosslink weak polyelectrolyte microcapsules containing carboxylic and amine groups, yielding stable structures.^35,36

The above mentioned techniques are exclusively based on a reaction between the functional groups of the two components in the multilayers. We recently demonstrated that the multilayer microcapsules assembled from PAH and PSS could be considerably stabilized by crosslinking only the PAH component with glutaraldehyde (GA).³⁷ After crosslinking by 2% GA for 2 h, an apparently thicker capsule wall was obtained with higher folds. No alteration of the macroscopic topology of the capsules was observed after incubation in 0.1 M NaOH for 24 h. The crosslinking can significantly improve the mechanical strength of the capsules to resist osmotic pressure induced invagination. Consequently, both the critical pressure and the elasticity modulus (680 MPa) of the capsule walls are doubled compared with that of the control. The crosslinking can also greatly lower the permeability of the capsule walls. Quantitative analysis revealed that the permeation coefficient for dextran (M_w ≈ 250 kDa) was reduced by a factor of 3 after crosslinking. We further applied this method to the poly(ethylenimine) (PEI) and PAA weak polyelectrolyte microcapsules.³⁸ The crosslinked microcapsules can maintain their macroscopic topology at extreme low or high pH, while reorganizing their microstructures to enable selective permeation or rejection of macromolecules at lower (pH<4) and higher pH (pH>6), respectively. Using this property, dextran with a molecular weight of 2000 kDa was successfully encapsulated. Thus, it is possible to produce capsules that are at the same time pH-responsive as well as stable over a large pH range.

Hydrogen bonding is the first non-electrostatic interaction used in theLbL assembly. More information about this topic can be found from a recent review paper and the references therein.³⁹ It is well known that the hydrogen bonded multilayers will disassemble at physiological conditions.^40,41 When they are used for biomedical applications, there is a need to stabilize the film too. The most commonly used method for crosslinking of the hydrogen bonded multilayers and capsules to date is the carbodiimide chemistry, which induces the formation of amide linkages.^42,43 For example, stability of the hydrogen bonded multilayer microcapsules is improved by the carbodiimide chemistry using ethylenediamine as a foreign crosslinking reagent for the poly(methacrylic acid) component.⁴⁴ The uncrosslinked component can be selectively released at elevated pH, yielding single component and hydrogel like microcapsules.^45,46 These capsules exhibit reversible pH-responsive swelling and shrinking, which can be used for loading and release of macromolecules. Revisable stabilization of hydrogen bonded LbL capsulesviadisulfide bonds has also been reported.^47,48 The crosslinked films are stable in neutral or alkaline solutions, but disassemble rapidly under reductive conditions.

2.2 LbL capsules based on other driving forces

It is known that different driving forces bring substantial differences to the microcapsules in terms of their chemical and physical structures, stability and stimuli-response, and inevitably their functionality and applicability. The driving forces for LbL assembly of microcapsules mainly began with electrostatic interaction,^4–6 which limits the building blocks to a narrow range of charged species. Then hydrogen bonding has been applied to synthesis of core–shell particles and hollow capsules as discussed above. Recently, the multilayer hollow microcapsules based on covalent bonding, base pair interactions, van de Waals interactions and guest–host interactions have been fabricated, which show unique properties.

The first example of covalent LbL assembled microcapsules was demonstrated by Xu and co-workers.⁴⁹N-Methyl-2-nitro-diphenylamine-4-diazoresin (NDR) and m-methylphenolformaldehyde resin (MPR) dissolved in methanol were alternately adsorbed on polystyrene (PS) microparticles through direct covalent reaction. Hollow capsules were obtained after removal of the PS particles. These capsules are stable enough to withstand the long etching time of strong polar organic solvents. The use of LbL strategy from aqueous systems to nonaqueous systems greatly widens the range of materials used for LbL assembly. We recently fabricated a new structure of microcapsules with high modulus and high stability through covalent LbL assembly (Fig. 2).⁵⁰Aminosilanized SiO₂ microparticles were used as templates. Poly(glycidyl methylacrylate) (PGMA) and PAH were alternately immobilized onto the particle surfaces through a coupling reaction between the epoxides and the amines. Thus, a highly crosslinked structure was produced in this process. When the desired layer number was reached, the particles were removed by HF solution, resulting in hollow microcapsules. The microcapsules are stable in extreme pHs and at elevated temperature. Using the method of osmotic-induced invagination,^31,32 the elastic modulus of the microcapsule walls without any treatments was found to be as high as 910 MPa. The acid- and base-treatment cannot decrease the modulus of the microcapsule walls.

Fig. 2 Schematic illustration of the process of direct covalent LbL assembly on a silica particle, and fabrication of a hollow capsule by etching out the template core. The blue lines represent PGMA, the red lines represent PAH, and the green dots represent the covalent linkage between layers. Reprinted with permission from ref. 50. Copyright 2007, Wiley-VCH.

Making use of the features of the reaction between amine and aldehyde, i.e. fast and efficient in aqueous solution at room temperature, we recently developed a method to fabricate single polyelectrolyte component multilayers and microcapsules through direct covalent assembly of PAH with a low molecular weight dialdehyde linker -GA.⁵¹ The structure as well as the cut-off molecular weight of the capsule walls can be tuned by the molecular weight of the polymer blocks.⁵² Because GA can readily react not only with amino groups but also with hydroxyl groups under very mild conditions, this method can be extended to other macromolecules, especially biomacromolecules such as polysaccharides, polypeptides, and proteins as well. Stimuli-responsive covalent LbL assembled microcapsules have been recently reported by Caruso and co-workers.⁵³ They used the click chemistry approach to form multilayer films⁵⁴ (Fig. 3) on particles and obtained hollow microcapsules after core removal. PAA grafted with a minor amount of either alkyne (PAA-Alk) or azide (PAA-Az) were alternately assembled on silica particles through the click reaction between the Alk and Az. Due to the nature of the weak polyelectrolyte PAA in the shells, the capsules can reversibly swell and shrink by up to 70% when subjected to acid–base pH cycling. These two methods can fabricate single polymer component LbL capsules, thus are helpful in correlating the structures and their properties.

Fig. 3 LbL assembly of multilayer films using click chemistry. Reprinted with permission from ref. 54. Copyright 2006, American Chemical Society.

Host–guest interaction is another type of driving force frequently employed in supramolecular chemistry. The complex usually forms between two determined molecules, typically exemplified by cyclodextrin (CD) and its guests.^55–57 A few attempts have been made to build LbL multilayer films using this interaction.^58–60 It is known that the host–guest interaction is readily mediated by the host and guest molecules with respect to their matching degree and concentration. If charge interaction is further introduced, multi-responsive microcapsules can thus be expected. According to this design, multilayer microcapsules were fabricated by using the interaction between β-cyclodextrin (β-CD) and ferrocene grafted to a weak polyelectrolyte PAH, which can further introduce charge interaction into the capsule walls (Fig. 4).⁶¹ The microcapsules that consist of PAH-g-β-CD and PAH-g-ferrocene indeed show multi-responsiveness to environmental stimuli. For example, they swell and shrink at low and high pH, respectively. Incubation in a salt or β-CD solution can also mediate their swelling and shrinking behavior. With these smart features, the microcapsules can serve as reservoirs for drugs, DNAs, enzymes and so on.

Fig. 4 LbL assembly of the same polyelectrolyte on carbonate particles to obtain hollow microcapsules using host–guest interactions. The chemical structure of PAH-g-β-CD, PAH-g-ferrocene, and β-CD/ferrocene inclusive are shown in the second row.

Assembly of DNA multilayer films both on planar substrates and colloidal particles from engineered sequences through specific base pair interaction has been demonstrated recently by Caruso’s group.^62–64 It was found that the composition and sequences of the oligonucleotides play an important role in the structure and assembly of the DNA multilayer films. After removal of the colloidal particles, hollow DNA capsules were obtained and their shrinkage could be controlled by the composition of the DNA sequences used for the multilayers’ assembly.⁶⁴ The shrinkage of the capsules can be used for encapsulation of desirable substances.⁶⁵

Akashi et al. recently reported the preparation of stable, ultrathin films of a double stranded poly(methyl methacrylate) (PMMA) stereocomplex by the alternate LbL assembly of it- and st-PMMA on a solid substrate (it denoted isotactic, st denotes syndiotactic).⁶⁶ Later on, the same group applied the it-/st-PMMA stereocomplex films onto silica particles and obtained hollow capsules of nonionic multilayers based on van der Waals interactions after core removal.⁶⁷ However, the specific properties correlated with the nature of the van der Waals interactions need further exploration.

3 Bio-related applications of multilayer microcapsules

3.1 Delivery carriers

For most of the potential applications of LbL microcapsules such as drug delivery, catalysis, cosmetics, biotechnology and nutrition, the controlled loading and release of a variety of substances is basically required. As aforementioned, since several review papers^17,20,21 have focused on this subject, we introduce here only the very recent progress of the related works.

3.1.1 Loading. Many techniques for loading the multilayer microcapsules with diverse substances have been developed so far, including permeability switch triggered by ionic strength,⁶⁸ pH,^69,70solvent polarity,⁷¹ and temperature,⁷² direct assembly of the multilayers on crystals or aggregates of the desired substances,^73–78 and pre-absorption of the desired substances into the porous templates^79–82 or pre-precipitation on the template surfaces^12,83 followed by LbL coating. Here we introduce two other newly developed methods: spontaneous deposition attracted by the preloaded substances^84–86 and coprecipitation of the substances into the template particles during particle fabrication.⁸⁷

The spontaneous deposition is based on a mechanism of high affinity of the preloaded substances with the substances of interest. This is initially driven by an interesting phenomenon that positively charged molecules such as dextran labeled with tetramethylrhodamine isothiocyanate (TRITC-dextran) can largely deposit into the aged “hollow” microcapsules templated on melamine formaldehyde (MF) particles.⁸⁵ By comparison of the fluorescence intensity from the capsule interiors to the capsule surroundings, it is obvious that a considerably high concentration of dextran in the capsule interiors has been achieved (the so-called spontaneous deposition) (Fig. 5a). Many other water-soluble substances with positive charges such as polyelectrolytes,⁸⁴proteins,⁸⁴ enzymes⁸⁸ and low molecular weight dyes and anti-cancer drugs (Fig. 5b, c)⁸⁵ can be spontaneously deposited with a large of quantity (the interior concentration is tens to more than hundred times higher than that of the bulk). Moreover, the deposition still occurs even if the molecules have very weak positive charge such as the TRITC-dextran (Fig. 5a), which is generally regarded as a neutral molecule but actually gains slight positive charges from a few pendent TRITC groups. The driving force for this phenomenon is attributed to the existence of a negatively charged complex (PSS/MF) within the capsule interior, which is formed by the dissociated PSS from the very initial layer and the positively charged MF degradation product (Fig. 6). It is worth noting that black rings emerged in between the capsule walls and the deposits (Fig. 5a, inset). This phenomenon is typical for spontaneous deposition, which might reflect the distribution of the negatively charged complex. The charge repulsion between the complex and the inner capsule wall (negatively charged PSS as the first layer) may lead to the floating of the complex in the capsule interior rather than attachment onto the capsule wall.

Fig. 5 (a) Fluorescence intensity averaged from inside the circles as a function of incubation time. TRITC-dextran (M_w ≈ 65 kDa) and preformed MF-(PSS/PAH)₅ capsules were used. (b) TEM images of Daunorubicin (DNR) deposited MF-(PSS/PAH)₅ capsules. (c) DNR and rhodamine B (RdB) concentrations in the capsule interior as a function of temperature. MF-(PSS/PAH)₄(PSS/PDADMAC)₅ capsules were used for DNR with a feeding concentration of 30 mg ml⁻¹, and MF-(PSS/PAH)₅ capsules for RdB, 80 mg ml⁻¹. The numbers in the figure represent the concentration ratios of the capsule interior and the bulk. PDADMAC = polydiallyldimethyl ammonium chloride. Reprinted with permission from ref. 85. Copyright 2005, Wiley-VCH.

Fig. 6 Schematic illustration to show the microstructure of the polyelectrolyte capsule containing a trace amount of negatively charged MF/PSS complex, by which positively charged DNR and RdB are sucked into the capsule interior to form a higher concentration than that of the bulk (the so-called spontaneous deposition). Reprinted with permission from ref. 85. Copyright 2005, Wiley-VCH.

By pre-filling the microcapsules with charged polyelectrolytes, loading of oppositely charged substances with a higher concentration in the capsule interiors can be similarly achieved. For example, due to the electrostatic interaction of positively charged doxorubicin hydrochloride (DOX) and negatively charged dextran sulfate, the loading amount of DOX increases about 3-fold when the capsules are filled with low molecular weight dextran sulfate relative to that of the hollow ones.⁸⁶

The coprecipitation method was first reported by Sukhorukov and co-workers.⁸⁷ A Na₂CO₃ solution was added to an equal volume of a CaCl₂ solution with the same concentration containing the macromolecules such as proteins and enzymes, which were then coprecipitated during the CaCO₃ microparticle growth. Core removal after LbL assembly of the multilayers leads to release of the macromolecules into the polyelectrolyte microcapsule interiors. The mild fabricating conditions can largely preserve the enzymatic activity of encapsulated α-chymotrypsin up to 85% by this method. The concentration of the loaded substances can be easily controlled by their initial concentrations in the mixed reaction solution, and much larger amounts of the macromolecules can be achieved. Moreover, a wide range of substances such as polyelectrolytes,^89,90 enzymes,⁹¹ DNAs,⁹¹polysaccharides⁹² and micelles⁹³ have been encapsulated by this method

More recently, we found that the capsules filled with PSS obtained by the coprecipitation method can effectively sieve charged molecules (Fig. 7).^89,90 While the microcapsules reject the permeation of negatively charged species, they can attract the deposition of positively charged molecules. The selectivity is very sensitive. For example, the capsules completely exclude dextran labeled with fluorescein isothiocyanate (FITC-dextran), but are permeable to TRITC-dextran having a similar molecular weight (from 4 kD to 70 kD), although there are only few charged dyes in a dextran chain.

Fig. 7 (a) Schematic illustration of the topology of PSS encapsulated microcapsules. (b) Scanning force microscopy (SFM) image of an intact capsule with free PSS trapped inside. Confocal laser scanning microscopy (CLSM) images of the PSS filled microcapsules incubated in solutions of (a) fluorescein for 2 h and (b) rhodamine 6 G for a few seconds. Reprinted with permission from ref. 89. Copyright 2005, American Chemical Society.

3.1.2 Release . There are two methods of desired release for the loaded substances: burst release and sustained release. The burst release is required when the capsules are internalized by cells to release the drugs or genes, or the capsules are targeted to cancer cells to release the anti-cancer drugs. On the other hand, sustained release is desirable when the capsules are proposed to remain in the blood circulation system to keep the effective drug level constant.

The most straightforward way to realize burst release is to destroy the capsule wall by external or internal stimuli. For example, some metal nanoparticles and dyes incorporated into the multilayer capsule walls can absorb near-infrared lasers to induce a local temperature increase and destroy the walls, leading to burst release.^94–98Ultrasonication is another way which can remotely destroy the capsule walls rapidly (in 5 s).^99,100 The presence of magnetite nanoparticles in the capsule walls can enable magnetically controlled delivery to the desired site and significantly improve the efficiency of the ultrasonically stimulated release of their content. It is notable that the encapsulated substances can be released at an ultrasonic power comparable to those applied in medicine, demonstrating the practical applicability of this novel method.

The decomposition of the capsule walls also can be triggered by specific chemical stimuli. For example, the capsules based on hydrogen bonding and crosslinked by disulfide bonds are stable at normal physiological conditions. The disulfide bond can be cleaved in a reducing environment, inducing the immediate deconstruction of the hydrogen bonded multilayer capsules and the release of loaded susbtances.^47,48 De Smedt and co-workers synthesized a polyelectrolyte containing phenylboronic acid as a glucose-sensitive moiety, with which multilayer microcapsules having glucose-sensitivity were assembled.¹⁰¹ In a solution containing 2.5 mg ml⁻¹ or 5 mg ml⁻¹glucose the capsules were dissolved within 5 min. Therefore, this kind of capsule is very promising for delivery of glucose related drugs such as insulin. The same group also reported the “self-exploding” microcapsule, which is composed of a biodegradable dextran gel core surrounded by a polyelectrolyte membrane.¹⁰² The gel can be hydrolyzed to yield products which are not permeable through the capsule wall. Consequently, when the osmotic pressure inside the capsule is high enough, the polyelectrolyte membrane is ruptured to release the loaded substances. Later on, the same group reported on degradable polyelectrolyte capsules containing one or two polyelectrolytes which can be degraded either enzymatically or through hydrolysis.⁹² After these capsules are internalized by cells, they will be subsequently degraded and release the encapsulated substances into the cells.

Sustained release of loaded materials can be accomplished by gradual decomposition of capsule walls or slow diffusion through capsule walls. For instance, biodegradable hollow capsules loaded with proteins have been prepared viaLbL assembly of chitosan and dextran sulfate on protein-entrapped mesoporous silica particles and the subsequent core removal.¹⁰³ In the presence of chitosanase, the chitosan component can be gradually degraded with time prolongation, and the capsules begin to deform and are finally destroyed. Thus the encapsulated proteins are released in a sustained manner and the release behavior can be manipulated by the chemical compositions of the capsule surface. The enzymatic deconstruction of LbL capsules also can be realized through incorporation of enzymes into their interiors. For example, Kreft and co-workers prepared self-disintegrating microcapsules by encapsulating a highly active mix of proteases into polypeptide LbL microcapsules (Fig. 8).⁹¹ Pronase was coprecipitated into CaCO₃ microparticles that were subsequently coated with poly(L-arginine)/poly(L-aspartic acid) multilayers. Then the enzyme was released into the capsule interiors and started to digest the capsule walls after core removal. The biggest advantage of this method is that by varying the amount of encapsulated pronase, lifetimes of such self-disintegrating capsules can be successfully adjusted from seconds and hours to days, thus the sustained release of the co-encapsulated DNA can be adjusted.

Fig. 8 Principle of time-delayed drug release from enzyme-degradable polyelectrolyte capsules. (A) Co-immobilization of a polyelectrolyte degrading enzyme and drug molecules in CaCO₃ microparticles. (B) LbL coating with specific, enzyme-degradable polyelectrolytes. (C) Core-dissolution leads to release of enzyme and drug into the inner void of the capsule. (D) Enzyme digests capsule-shell and releases drug. Reprinted with permission from ref. 91. Copyright 2007, Wiley-VCH.

The spontaneously deposited low molecular weight drugs in the LbL microcapsules templated on MF colloidal particles can be sustainably released by diffusion through the capsule walls.^85,104 The amount of loaded drugs is proportional to the drug feeding concentration, temperature and salt concentration, demonstrating tailorable deposition behavior that is crucial for the drug delivery vehicle. The deposited drugs could be released again in a sustained manner and their behaviors can be manipulated by changing the interaction between the drugs and the PSS/MF complex existing in the hollow capsules by ionic strength, pH and temperature. The presence of anti-cancer drug loaded capsules was seen to steadily decrease the cytoviability of HL-60 cell line, a kind of human leukemia cell.⁸⁵

For better control of the spontaneous deposition properties, we further used the coprecipitation method to load the microcapsules with polyelectrolytes.^{89,90,105–107} Quantification of the anti-cancer drugs of daunorubicin (DNR) and DOX loading was performed under different conditions, revealing that a larger amount of drugs could be incorporated at higher drug feeding concentrations and higher salt concentrations.^105,106 They showed a strong ability to accumulate the positively charged DOX with a factor of tens to hundreds, that is, the drug concentration within the microcapsules was hundreds of times higher than the feeding concentration. The drug release behaviors from the microcapsules with different layer numbers were studied too, revealing a diffusion controlled release mechanism at the initial stage (4 h). Then application of the drug loaded capsules for tumor treatment was demonstrated by in vitrocell culture and in vivo animal experiments. The in vitro experiments showed that the encapsulated drug can effectively induce the apoptosis of HepG2 tumor cells. By seeding the HepG2 hepatoma cells into nude mice, tumors were created for the experimental studies. The results showed that the encapsulated DOX has better efficacy than that of the free drug in terms of tumor inhibition in a 4 week in vivo culture period.¹⁰⁷

Besides from the capsule interiors, release of biologically relevant substances such as DNA, proteins and enzymes from the shells of the capsules and core–shell particles can also be achieved by disassembly or degradation of the shells, such as presented by Donath and co-workers.¹⁰⁸ They coated silica particles with protamine and dextran sulfate multilayers, in which plasmid DNAs were embedded. The particles could be taken up by HEK 293 T cells, and then the multilayers were defoliated to release the DNA, resulting in the expression of plasmid encoded proteins in the cells. An interesting point is that different plasmids can be incorporated to a different depth in the multilayer shells, thus co-expression of different proteins in a controlled manner is possible. Moreover, the tailor-made LbL capsules or core–shell particles may have both protective function and targeted delivery properties. In a later work,¹⁰⁹ the same group used the capsules encapsulated with pH sensitive fluorescent polymer conjugates to report the intracellular environment pH of the internalized colloidal particles. They identified endosomal escape as the bottleneck limiting the efficacy of the transfection.

3.2 Surface modification for stealth function or targeting

Surface properties greatly influence the performance of microcapsules in the biological environment. Thus considerable efforts have been given to surface modification of microcapsules and multilayer coated colloids so that they will not be recognized by the hosts as foreign materials or they can be targeted to specific sites or cells. More details on fabrication of LbL capsules with biocompatible surfaces or decorated with biofunctional molecules can be found in recent reviews.^21,26 Below we mainly summarize the more recent progress in this direction.

It has been verified that the assembly of a final monolayer of polycationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) onto the PSS-terminated multilayers can impart protein resistance to the surface.¹¹⁰ The quality of the PLL-g-PEG layer, which greatly influences the protein resistance of the surface, on the LbL microcapsules depends on both the type of core material and the dissolution protocols used. The best protein resistance is achieved by using poly(lactic acid) (PLA) cores and coating the microcapsules with PLL-g-PEG after core removal. If the top copolymer layer contains a fraction of the PEG chains end-functionalized with biotin, it may further specifically recognize and immobilize controlled amounts of streptavidin on the capsule surfaces. These capsules have potential applications as novel platforms for biotechnological applications such as biosensors and carriers for targeted drug delivery. One of the other important requirements for biomedical applications is the escape of clearance by the mononuclear phagocytic system (MPS). Merkle and co-workers further studied the effects of PLL-g-PEG and poly-L-glutamic acid (PGA)-g-PEG coating on the PAH/PSS capsules in terms of MPS recognition through in vitrocell culture of human monocyte derived dendritic cells and macrophages.¹¹¹ It was found that the PGA-g-PEG coating had no significant effect on the cellular recognition, possibly due to the insufficient PEG density. In contrast, PLL-g-PEG effectively blocked the phagocytosis of the coated microcapsules (Fig. 9).

Fig. 9 Phagocytosis of coated microcapsules: dendritic cells (left) and macrophages (right) were incubated with 4.8 μm (left) or 1.2 μm (right) microcapsules for 4 h. Images were obtained by an overlay of phase contrast and fluorescence channels. (A, B) PSS-coated microcapsules; (C, D) PLL-coated microcapsules; (E, F) PLL-g-PEG coated microcapsules. Scale bars = 50μm. Reprinted with permission from ref. 111. Copyright 2008, American Chemical Society.

Nowadays great interest has been attracted by integration specific biofunctionality onto the capsule surface for targeting. The concept has been demonstrated recently by targeting the microcapsules on a patterned surface by electrostatic interaction^112–114 or recognition between biotin and avidin.¹¹⁵ By microcontact printing, patterns of oppositely charged polyelectrolytes to the top layer of the substrate multilayers were created, which were then used to attract the microcapsules by electrostatic interaction, forming capsule arrays.¹¹⁴ Moreover, a method that allows isolation of the individual capsules and the patterning assembly with satisfactory spatial selectivity is developed via avidin–biotin recognition (Fig. 10).¹¹⁵ The stable microcapsule arrays were further utilized as microreactors to synthesize quantum dots (QDs) and other nanoparticles. Release of the spatially synthesized products can be tuned easily as well (Fig. 10). Donath and co-workers recently demonstrated that the phosphatidylserine coated LbL core–shell microparticles or capsules fused with rubella-like particles (RLPs) can facilitate the membrane passage (Fig. 11).¹¹⁶ On incubation of the lipid-coated LbL colloids with RLPs at low pH (pH 4), the particles attached to the lipid layer by electrostatic forces and subsequently fused with the membrane, presenting the virus envelope proteins on the particle surface. The key function of the virus surface is the binding to a host cell surface, induction of endocytosis, and subsequent fusion with the endosome membranes. Cell culture results confirmed that the particles integrated with RLPs can retain their biological activity and enhance the cell membrane passage.

Fig. 10 (a) Schematic illustration to show the strategy and bio-affinity force for capsule patterning. Avidin molecules are covalently patterned on a polymer film having pentafluorophenyl esters by microcontact printing, which are used to guide the spatial location of biotinylated capsules. Polymers with chelating groups (exemplified with PVA here) are loaded in the capsules to facilitate precipitation or reduction reactions to synthesize QDs, nanocrystals and nanoparticles (illustrated by the production of the ZnS QDs). (b) Fluorescence image to show the existence of ZnS QDs formed exclusively within the aligned microcapsules. (c) Excitation and emission spectra of aligned microcapsules containing ZnS QDs. (d) The evolution of the fluorescence emission at 470 nm as a function of time. The solid and the dashed lines represent the fluorescence intensity from the as-prepared QDs containing capsule arrays and aligned QDs containing capsules having additional (PSS/PAH)₃ layers, respectively. Insets show the fluorescence images of capsule patterns after incubation in buffer for 36 d. The scale bars in (b) and (d) represent 30 μm and 20 μm, respectively. Reprinted with permission from ref. 115. Copyright 2006, Wiley-VCH.

Fig. 11 Protocol for engineering virus functionality on polyelectrolyte lipid composite colloids and capsules. Removal of the core is optional. Reprinted with permission from ref. 116. Copyright 2005, Wiley-VCH.

Targeting and uptake of specific antibody modified LbL particles and capsules to colorectal cancer cells were recently demonstrated by Caruso and co-workers.¹¹⁷ They immobilized the humanized A33 monoclonal antibody (HuA33 mAb) which can bind to a transmembrane glycoprotein, the human A33 antigen, on the LbL particles and capsules through electrostatic interaction. A33 antigen is expressed by 95% of human colorectal tumor cells as well as on the basolateral surfaces of intestinal epithelial cells, but not by other epithelial tissues. The cell culture experiments confirmed that the HuA33 mAb decorated particles can be targeted and selectively internalized by colorectal tumor cells and the binding was found through antibody–antigen specific interaction.

3.3 Biosensors

Because of the diverse loading methods for assay elements and easy functionalization, the LbL microcapsules and microparticles are one of the ideal platforms for sensing applications. McShane’s group contributes significantly to this area. In their earlier work,¹¹⁸ the capsules were loaded with a model fluorescent assay consisting of a sodium-sensitive dye and a reference fluorophore. The capsules are mechanically robust, and the encapsulated fluorophores are well retained. Results from sodium sensitivity experiments suggest that the capsules have an excellent potential for use as sensors, with a highly linear response over a broad range of concentrations (0–100 mM). Later on, an oxygen sensor was designed using a similar strategy.¹¹⁹ Besides the fluorescent assay, the microcapsules are also attractive for encapsulating competitive binding assays that require free movement of molecules inside the capsules.¹²⁰

Hydrogels have several unique properties that facilitate the loading of a variety of enzymes and dyes for chemical sensing. However, control over release rates or stable encapsulation is still a challenge. Recently the McShane group designed a glucose sensor by encapsulation of glucose oxidase (GOD) and an oxygen-quenched ruthenium compound within calcium alginate microspheres followed by multilayer coating.¹²¹ The multilayer films were used to stabilize the entrapped enzyme and control substrate diffusion. It was demonstrated that the application of multilayer thin films to the alginate microspheres was effective in reducing the leaching rate and total loss of the encapsulated materials from the microspheres.^121,122 The polyelectrolyte pairs for assembly, layer number and crosslinking all have great influences on the loss rate of the encapsulated enzyme and the enzyme activity for long term storage.^123,124 These findings demonstrate that enzyme immobilization and stabilization can be achieved by using simple modifications to the LbL assembly technique.

The LbL multilayer microcapsules also can work as mobile pH-sensors to monitor the local pH inside living cells (Fig. 12).¹²⁵ The microcapsules were loaded with a pH sensitive, high molecular weight SNARF-1-dextran conjugate. SNARF-1 exhibits a significant pH-dependent emission shift from green to red fluorescence under acidic and basic conditions, respectively. Its unique spectral properties are maintained after the encapsulation as well. Therefore, the pH change of the local environments of the SNARF-1-filled capsules can be monitored during the transition from the alkaline cell culture medium to the acidic endosomal/lysosomal compartments of human breast cancer cells and fibroblasts.

Fig. 12 SNARF-loaded capsules change from red to green fluorescence upon internalization by MDA-MB435S breast cancer cells. (A) SNARF-fluorescence after adding the capsules to the cell culture and 30 min equilibration. Most of the capsules are outside of the cells and exhibit red fluorescence due to the alkaline pH of the medium. (B) The same cells after another 30 min of incubation. Capsules remaining in the cell medium retain their red fluorescence (red arrows). Capsules that were already incorporated in the acidic endosome in the first image retain their green fluorescence (green arrows). Some capsules were incorporated in endosomal/lysosomal compartments inside cells within the period of 30 min, which is indicated by their change in fluorescence from red to green (red to green arrows). Both images comprise an overlay of microscopy images obtained with phase contrast, a red and a green filter set. (C) Schematic presentation of the endocytotic capsule uptake. Reprinted with permission from ref. 125. Copyright 2007, Royal Society of Chemistry.

The multilayer capsules loaded with low molecular weight fluorescent compounds have also been utilized in immunoassays, which has 70–2000 fold higher sensitivity than that of the corresponding immunoassay performed with direct fluorescently labeled antibodies.¹²⁶ Donath and co-workers recently used the LbL approach to obtain microbeads coated with authentic viral surfaces, which can catch and quantify virus-specific antibodies in a flow cytometric analysis.¹²⁷ The antigenic surfaces were generated by fusion of multiple virus subtypes to a lipid bilayer, which surrounds the multilayer coated particles. The multilayer thin film can even be used to coat the living mammalian cells as a protective shell, which allows free diffusion of small molecules, but prevents internalization of proteins larger than 60 kDa into the cells.¹²⁸ The best cell survival after coating is as high as >80% and the coated cells can be used as biosensors to detect specific compounds.

3.4 Bioreactors

3.4.1 Enzyme-catalyzed biomineralization. Sukhorukov and co-workers loaded PAH/PSS capsules with urease through a solvent polarity induced permeability switch.⁷¹ When these capsules are dispersed in a water solution containing CaCl₂ and urea, the urease inside catalyzes the decomposition of urea, yielding carbonate anions exclusively inside the capsules where they react with Ca ions to form CaCO₃ (Fig. 13). The reaction will stop when the CaCO₃ completely fills the capsule volume. Surprisingly, only the metastable vaterite phase of CaCO₃ was detected, while the more stable calcite phase was not.¹²⁹ Using a similar strategy, biocompatible polypeptide capsules with high enzyme loading and activity prepared by templating on mesoporous silica spheres have also been used as biomimetic reactors for CaCO₃ synthesis exclusively inside the capsules.¹³⁰

Fig. 13 Optical microscopy images of CaCO₃growth at different stages. Reprinted with permission from ref. 129. Copyright 2003, Wiley-VCH.

3.4.2 Enzyme-catalyzed polymerization. Besides the inorganic particles, polymers also can be polymerized exclusively within the microcapsules under enzyme-catalyzation, as demonstrated by Lvov and co-workers.¹³¹ This approach is based on selective permeability of the capsule walls for monomers, while the biocatalysts and the formed polymers cannot diffuse out of the capsules. Horseradish peroxidase (POD) was encapsulated into the PSS/PAH capsules through a pH induced permeability switch.^69,704-(2-Aminoethyl) phenol hydrochloride (tyramine) was used as a monomer which can easily diffuse into capsule interiors and emit easily detectable fluoresce after polymerization. It was found that the capsules were filled with polymers and their amount could be controlled by the initial monomer concentration. The same group further demonstrated that biocatalytic polymer synthesis could occur on the LbL microcapsule surface.¹³² The POD was first assembled onto the capsule walls by alternate adsorption with polyelectrolytes with preserved activity for enzyme-catalyzed polymerization of 4-oxyphenols. Using an outermost surface POD layer as a template and varying the phenol type, buffer pH, and reaction component concentrations, the thickness of the phenolic polymer coating on the capsules can be controlled within a range of 20–50 nm.

3.4.3 Bienzyme reaction. The polyelectrolyte microcapsules have also been used to design bienzyme systems. This was achieved by loading POD and GOD into the capsules templated on MF particles followed by the partial decomposition of the cores.¹³³Oxidation of glucose by GOD leads to the formation of H₂O₂, which, in the presence of amplex red, acts as a substrate for POD. Amplex red is then converted into the highly fluorescent resorufin by POD. This bienzyme system loaded into the microcapsules was proved suitable for kinetic glucose content assay and was more stable than the enzyme system in the aqueous solution. In a similar work,¹³⁴GOD and POD were coprecipitated with CaCO₃ microspheres, followed by LbL assembly of multilayers on their surfaces to obtain microcapsules. Due to the substrate diffusion limitation by the capsule walls, an 85% decrease in the rate of glucose consumption relative to GOD and POD in free solution was observed. Confocal imaging, which can monitor the microscale glucose consumption, revealed that the high concentration of enzyme immobilized within the capsule walls resulted in a greater rate and quantity of glucose consumption near the inner surface of the capsule walls in comparison with that of the capsule interiors.

LbL technology is especially convenient for fabricating complex structures, such as shell-in-shell structures.¹³⁵ Recently, LbL microcapsules with a shell-in-shell structure were fabricated for integrated and spatially confined enzymatic reactions (Fig. 14).¹³⁶ The strategy involves the fabrication of spherical ball-in-ball particles consisting of two concentric CaCO₃ compartments that can be independently loaded with macromolecules. These two compartments are separated by the polyelectrolyte multilayers. By further deposition of the polyelectrolyte multilayers on the surfaces of ball-in-ball particles followed by decomposition of the CaCO₃ particles, novel shell-in-shell microcapsules containing biomacromolecules separated by semipermeable membranes can be obtained. GOD and POD were encapsulated in the outer and inner compartments, respectively. The coupling of both enzymatic reactions could be mediated by the semipermeable character of the polyelectrolyte shells, which allow small substrate (and product) molecules to diffuse in and out, whilst confining the enzyme molecules in the isolated compartments. When glucose is added, H₂O₂ is generated in the outer compartment, and diffuses into both the bulk solution and the inner compartment. When amplex red is added, resorufin is only formed in the inner compartment as both POD and H₂O₂ are required. One can observe that the fluorescence emission from the resorufin appeared within a few seconds in the inner compartment, which then spread into the outer compartment of the shell-in-shell structure (Fig. 15). Such two compartment capsules exhibit exciting potential especially for biomedical reactions in a confined space. The barriers between the integrated components can be remotely removed, thus enabling mixing and, hence, the start of a reaction by an external trigger. This was demonstrated later by the same group.¹³⁷ Light absorbing gold nanoparticles were incorporated into the inner shells. The intermixing of the contents of the two compartments occurred when the inner shells were disrupted by a near-infrared laser light illumination.^94–98

Fig. 14 General route for the synthesis of shell-in-shell microcapsules. A = initial core; B = core–shell particle; C = ball-in-ball particle (type I); D = ball-in-ball particle (type II); E = shell-in-shell microcapsule. Reprinted with permission from ref. 136. Copyright 2007, Wiley-VCH.

Fig. 15 Coupled enzymatic test using GOD and POD inside shell-in-shell capsules. (a) Reaction schemes. (b) Localization of GOD and POD within shell-in-shell capsules (c) CLSMin situ imaging of resorufin formation. Reprinted with permission from ref. 136. Copyright 2007, Wiley-VCH.

3.4.4 ATP synthesis . The polyelectrolyte multilayer capsules with tailored structures and functionalities are ideal model systems as artificial cells. The adsorption of a lipid bilayer onto the polyelectrolyte multilayer coated colloids or microcapsules is the first step towards mimicking the cell membrane.^9,138–141 By integration of ion channel-forming peptides into the lipid bilayer, different ions can be selectively transported through the capsule walls.¹⁴² Li and co-workers recently demonstrated the integration of chloroplastic F₀F₁ (CF₀F₁) ATPase into a microcapsule wall containing a lipid bilayer and synthesis of adenosine triphosphate (ATP) by this composite capsule (Fig. 16).¹⁴³ The Lipid bilayer decorated microcapsules with incorporated CF₀F₁ATPase were obtained by a combination of the CF₀F₁ proteoliposomes and the microcapsules, which allows the proteolipid to adsorb favorably on the outer shells of the microcapsules through electrostatic interaction. The driving force for ATP synthesis catalyzed by CF₀F₁ATPase is provided by an acid/base transition between the interiors and exteriors of the capsules. To realize this, buffer solutions with pH values of 8.8 and 4.6 were successively added to create a proton gradient between the inside and outside of the capsules. A pH sensitive fluorescent probe was filled into the capsules to detect the proton transportation across the membrane through CF₀F₁ATPase during the synthesis of ATP, which corresponds to the activity of ATPase. In the first 300 s of reaction, the light intensity decreases with the time elapsed, after which it slowly approaches a constant value, indicating that proton transportation occurs across the capsule walls through CF₀F₁ATPase from the exterior solution to the interior solution due to the proton gradient. As the proton gradient vanishes inside and outside the capsule solution, the ATP synthesis reaction stops. The measurement of ATP concentration confirmed that the ATP amount in the capsule suspension continuously increases over the reaction period and is obviously higher after addition of Triton X–100, which can enhance the release of ATP from the interior of the capsules (Fig. 17). These results indicate that ATP has been synthesized inside the microcapsules. The same group further assembled CF₀F₁-ATPase on lipid-coated hemoglobin protein capsules.¹⁴⁴ATP has been synthesized in the protein microcapsules by utilizing proton gradients produced from the oxidation and hydrolysis of glucose catalyzed by GOD.

Fig. 16 Schematic representation of the arrangement of CF₀F₁ATPase in lipid-coated microcapsules. ADP: adenosine diphosphate; Pi: inorganic phosphate. Reprinted with permission from ref. 143. Copyright 2007, Wiley-VCH.

Fig. 17 ATP biosynthesis as a function of reaction time in CF₀F₁/lipid-modified polyelectrolyte microcapsules (a) in the interior and exterior solutions after addition of 0.1% Triton X-100 and (b) in the exterior solution. Reprinted with permission from ref. 143. Copyright 2007, Wiley-VCH.

4. Conclusions and outlooks

The LbL assembled multilayer microcapsules present a class of advanced materials and have potential applications in many areas of science and technology, owing to their tailorable structures, compositions and properties, and the ease of property manipulation and functionalization. In particular, recent attentions has been paid to the multilayer microcapsules assembled based on new driving forces, and the integration of biofunctional moieties into the microcapsules aiming at diverse bio-related applications, as illustrated in this review.

Although a lot of significant achievements have been made in this area in the past decade, there are still some challenges that need to be fulfilled. First, it is important to develop new strategies for the fabrication of LbL microcapsules or manipulation of their properties, with special purposes for functionalization and thereby applications. Different strategies can bring substantial differences to the microcapsules in terms of their chemical and physical structures, stability and stimuli-response, and inevitable their functionality and applicability. Moreover, former basic scientific researches on LbL capsules, most of which are based on electrostatic interaction and hydrogen bonding, focus on understanding their physicochemical properties, such as permeability and mechanical properties. Now works on mimicking the functionalities of cells such as biochemical reactions in a confined space and selective ion transportation are emerging, yet still more efforts should be given to this direction. For example, the LbL microcapsules mimicking the deformability of red blood cells by shape design and interior and surface engineering would be very helpful for drug delivery. Finally, drug delivery vehicles are one of the most possible applications of the LbL microcapsule so far, as revealed by those many existing studies. However, in our opinion, there are still many challenges remaining, including the application of cheaper raw materials and improvement of the fabrication efficiency, optimization of the drug release profile for a specific therapy or disease treatment, the integration of biofunctional moieties onto the microcapsule surfaces aiming at target delivery, and biocompatibility in both the tissue and blood contacting levels. For example, when the drug loaded microcapsules are used by a manner of vein injection, the microcapsule system must possess good enough blood compatibility for purposes of long term circulation and target delivery. For other applications, both the capsule structure and performance should be designed and controlled according to the requirements of the exact situation, and can compete with other existing methods. We believe these challenges will be addressed sooner or later, and can be expedited by collaborations of multidisciplinary investigators from chemistry, pharmaceutics, biology, material science as well as medicine.

Acknowledgements

We appreciate the financial support of the China Postdoctoral Science Foundation (No. 20070421171), Natural Science Foundation of China (No. 20434030 and 20774084) and the National Science Fund for Distinguished Young Scholars of China (No. 50425311).

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Footnote

† This paper is part of a Journal of Materials Chemistry theme issue on Biology in the Service of Materials. Guest editor: Vincent Rotello.

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