This journal is c The Royal Society of Chemistry 2010 Chem. Commun.

Cite this: DOI: 10.1039/c0cc04820h

Anisotropic multicompartment micro- and nano-capsules produced

via embedding into biocompatible PLL/HA filmsw

Mihaela Delcea,*aNarayanan Madaboosi,

aAlexey M. Yashchenok,

aPrabal Subedi,

a

Dmitry V. Volodkin,aBruno G. De Geest,

bHelmuth Mohwald

aand Andre G. Skirtach

a

Received 5th November 2010, Accepted 1st December 2010

DOI: 10.1039/c0cc04820h

We present a novel strategy to fabricate anisotropic multi-

compartment Janus capsules by embedding larger containers

into a soft poly-L-lysine/hyaluronic acid (PLL/HA) polymeric

film, followed by adsorption of smaller containers on top of their

unmasked surface. This research is also attractive for developing

substrates for cell cultures.

Novel and advanced biomedical applications of polymeric

microcapsules necessitate development of drug delivery

vehicles with increased degree of complexity and more

elaborate functionalities. In this regard, anisotropic

polymeric micro- and nano-capsules are an emerging trend1

in the field of capsules.2 Indeed, as it was highlighted by de

Gennes in his Nobel lecture in 1991,3 asymmetric particles offer

unique properties that are impossible to attain with

homogeneous or symmetric materials. As such, synthesis of

anisotropic structures is gathering increased interest due to

possibilities to mimic bio-molecular behavior.4

Synthesis of anisotropic polymer particles has been

of interest in macromolecular science for decades5 and

various strategies to develop anisotropic particles have been

already described. One promising approach involves the

immobilization of isotropic particles followed by selective

surface modification.6–8 In order to achieve that, different

methods can be used: (i) masking or trapping of one

hemisphere during surface modification;7,9 (ii) microcontact

printing;8,10 and (iii) use of fluxes or fields involving sputtering

of thin films.11 Due to their composition and morphology,

anisotropic capsules can be used for direction-specific release

which, in context of intracellular delivery, can serve for

targeting specific cells and even their sub-compartments.

Janus particles12 and capsules10 including those made by

fusion13 have been previously presented. However, to the

best of our knowledge, microcapsules anisotropic in

morphology and anisotropic multicompartment Janus micro-

capsules have not been reported. Besides, biocompatibility of

masking and materials used in production of anisotropic con-

structs remains an issue in previously reported studies.

Here we present a novel method for synthesizing anisotropic,

in morphology, multicompartment Janus capsules and

particles using biocompatible poly(L-lysine)/hyaluronic acid

(PLL/HA)12/PLL films for masking. The method relies on

partially embedding larger containers, coated particles or

capsules onto biocompatible (PLL/HA)12/PLL polymeric

multilayer films followed by the addition of smaller particles

to their outer unmasked surface, Fig. 1. Based on electrostatic

interactions, cationic poly(diallyldimethylammonium chloride)

(PDADMAC) and anionic poly(styrene sulfonate) (PSS)

were alternately deposited to form polyelectrolyte multilayers

(PEM) on the surface of large (4.8 mm) silica particles

(PDADMAC/PSS)4 and (PDADMAC/PSS)3PDADMAC on

small SiO2 (0.5 mm). Unlike thin linearly grown films (for

example, those composed of PSS and PDADMAC), which are

much thinner and which exhibit both strong inter-polymer

interaction and low polymer mobility, exponentially grown

biocompatible (PLL/HA)12 multilayer films have thicknesses in

Fig. 1 Schematics of anisotropic construct formation depicting the

following steps: (1) entrapping large silica capsules/particles (grey

spheres) in biocompatible polymeric (PLL/HA)12/PLL films (labeled

in green), followed by (2) adsorption of small silica containers (in red),

and (3) film destruction by NaOH and anisotropic construct extraction

onto a glass substrate.

aMax-Planck Institute of Colloids and Interfaces, 14424-Potsdam,Germany. E-mail: mihaela.delcea@mpikg.mpg.de

b Laboratory of Pharmaceutical Technology, Ghent University,9000-Ghent, Belgium

w Electronic supplementary information (ESI) available: Experimentaldetails, CLSM images of big and small particles, images of polymericfilms with embedded particles, images of isotropic particles before andafter treatment with NaOH, images of anisotropic capsules. See DOI:10.1039/c0cc04820h

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Chem. Commun. This journal is c The Royal Society of Chemistry 2010

the micrometre range (approximately 1 mm thick)14 due to ‘‘in’’

and ‘‘out’’ polymer diffusion.15 Their architecture, biological,

physico-chemical and mechanical properties are of great

relevance for cell culturing.15 The polymer diffusion in such

exponentially grown films allows for high accumulation of

material of interest on the film surface. Desorption of

polymers from the exponentially growing HA/PLL films is

considered to be their important functionality that can be used

for functionalization of films with, for example, nanoparticles.16

These films possess high loading capacity as a result of polymer

doping onto the film surface that results in the accumulation of

a large amount of adsorbing material (macromolecules or

nanoparticles), which is many times less for films possessing

lower polymer mobility.17,18 Similar PLL/HA films were

functionalized with DNA, nanoparticles, microcapsules, and

liposomes.17,19,20

There are three steps in our procedure to produce

anisotropic multicompartment constructs. Firstly, the anionic

large polyelectrolyte coated SiO2 particles/capsules were

embedded in (PLL/HA)12/PLL films by incubation; this is

shown in step 1, Fig. 1. Here, particles and capsules sediment

on the surface of the films due to gravity. This is a significant

step in the process because it governs further assembly.

Secondly, the films with embedded containers were

functionalized with smaller (0.5 mm) polyelectrolyte coated

SiO2 nano-containers (Fig. S3, ESIw), step 2 in Fig. 1.

Different concentrations of smaller SiO2 containers have

been used for adsorption, 0.5 mg mL�1 being the optimum.

Thirdly, the film was flipped upside-down and anisotropic

constructs were extracted by adding NaOH solution of

higher pH, which loosens the interaction of constructs with

films but does not affect anisotropic constructs (produced of

strong polyelectrolytes).

An important step of this process is loosening of the

interaction of anisotropic constructs with the films upon

their extraction. The strength of interaction can be decreased

by using a solution of high pH, for example NaOH.

Detachment of anisotropic constructs was preceded by a

control experiment, in which NaOH (at pH E 9) was added

to a solution containing capsules. This experiment showed that

smaller containers are well-attached to the larger ones after

addition of NaOH (Fig. S4, ESIw). At the same time, this

control demonstrates that adsorption of smaller containers

onto the bigger containers without masking leads to

the formation of isotropic structures. By adjusting the

concentration of NaOH one can affect detachment of

anisotropic constructs, and, eventually, destruction of the

film. For example, at a concentration of 0.01 M NaOH,

desorption and release of anisotropic constructs occurs

without film destruction. In such a way, the extraction of the

anisotropic particles/capsules is controlled and the aggregation

of anisotropic constructs is minimized. That leads to

possibilities to reuse the films for further immobilization of

particles/capsules. On the other hand, the film can be even

completely destroyed by adding 0.1 M of NaOH.

Concentration of initially embedded larger containers

determines the overall yield of anisotropic constructs; it

also affects aggregation. Therefore, we studied the influence

of concentration and found that 0.5 mg mL�1 of larger

containers is the optimal value (Fig. S1, ESIw); higher

concentrations lead to aggregation of anisotropic constructs

upon extraction, while lower concentrations reduce the overall

yield. The understanding of the kinetics of particle embedding

is essential to evaluate their interaction with the film;

our studies showed that 2 minutes was sufficient for

sedimentation of larger particles (Fig. S2, ESIw). In the case

of microcapsule embedding, the time sufficient for embedding

was found to be 10 min. The CLSM (Confocal Laser Scanning

Microscope) profile, Fig. 2(a), shows that larger containers are

embedded to h E 45% of their diameter, Fig. 2(b). That

leaves the upper half of the larger container available for

functionalization.

The anisotropic constructs were collected and imaged by

CLSM, Fig. 3. It can be noted that two approaches to

fabrication of anisotropic multicompartment capsules can be

pursued. In the first approach, a desired configuration of

particles is obtained and further processed into capsules.

Alternatively, direct embedding of microcapsules into

the film and their further functionalization with small

Fig. 2 (a) CLSM top view image of silica containers (5 minutes after

addition) partially embedded in a biocompatible (PLL/HA)12 film

doped with PLL-FITC (green). (b) The fluorescence profile, which is

taken across a particle embedded in the film, and which yields the

distribution of polymer around the particle; h is the protrusion.

Fig. 3 (a) Overlay (left-hand) and fluorescence (right-hand) images of

anisotropic particles obtained after destruction of the polymeric films.

Fluorescence images of anisotropic particle (b) and capsule (c). Smaller

nano containers are labeled in red, the yellow–green signal results from

the mixture of encapsulated dextrane Alexa-fluor 488 (green

fluorescence) and smaller containers (red). The white arrows indicate

the masked regions. The scale bars correspond to 20 mm in (a) and 3 mmin (b) and (c).

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This journal is c The Royal Society of Chemistry 2010 Chem. Commun.

nanocontainers can be used, Fig. 3(c) and Fig. S5 (ESIw).There, larger microcapsules loaded with Alexa-fluor

488 dextran (green) are functionalized with smaller SiO2

nano-containers with PAH-TRITC (red). Although diffusion

of molecules into the films17 poses certain limitation for

retaining the masking, the soft ‘‘hydrogel’’-like nature of

the multilayers combined with controllable thickness, high

loading capacity and polymer diffusion present significant

advantages21 of their application in cells. Furthermore,

controllable self-embedding and efficient recovery of

anisotropic structures open vast opportunities for diverse

biomedical applications as they can be loaded with different

bio-molecules and used as reactors for enzyme-catalyzed

reactions,22 mechano-biology23 and other applications.24 On

the other hand, functionalization of films with capsules and

control over their interaction is of interest for coatings as well

as substrates for cellular cultures.

In summary, we have presented anisotropic particles and

capsules produced by embedding and partially masking larger

containers into soft, biocompatible PLL/HA polymeric films.

This step is followed by adsorption of smaller containers from

the top onto the unmasked surface of larger containers.

Flipping the films upside-down and adding a solution of

NaOH at high pH (pH E 9) allows extraction of anisotropic

constructs by loosening their interaction with films; also,

this approach minimizes potential aggregation. The approach

presented here is versatile, applicable to a multitude of

particles and templates of various sizes and allows for

diverse functionalization. Various anisotropic structures can

be obtained by embedding capsules or particles into polymeric

films and by their further functionalization with particles

and/or capsules. Site-dependent loading and release of

macromolecules to/from such anisotropic containers will be

explored in our forthcoming studies.

Notes and references

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