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CXXXX American Chemical Society

Externally Triggered Dual Functionof Complex MicrocapsulesQiangying Yi and Gleb B. Sukhorukov*

School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS United Kingdom

Vesicles carrying active substanceswhich can enable response to multi-ple external stimuli have been widely

studied and developed as attractive candi-dates for delivery systems, micro/nanoreac-tors, and sensors, etc.1,2 One of the mostsuccessful approaches to tailor various func-tions in one entity is layer-by-layer (LbL)assembly, which provides an optional tech-nique to engineer a series of functional cap-sules due to its simplicity and versatility.3 Inparticular, a delivery system based on LbLcapsules has attracted increasing interestduring the past decades for its promisingapplications in different fields ranging frombiotechnology and pharmaceutics to che-mical synthesis and catalysis perspectives.4�6

Generally speaking, different external sti-muli have been employed to realize thefunctionalities of fabricated LbL capsulesystems, to which functional layers of re-sponsive polymers or nanoparticles are in-troduced, in order to meet their variousrequirements for capsules to respond totriggering stimuli, including remotely.1,7

Typically, light-addressable capsules repre-sent a series of the fast developed stimuli-responsive vesicles for potential use, as their

micro/nanostructures can be simply ad-justed by the external light. Optical treat-ment allows the functionalization of thesefabricated capsules with accompaniedchanges in their morphologies, shell stabi-lity, as well as permeability, benefiting fromunderlying chemical transitions.8�10

Previous investigations have developedlight-responsive capsule systems for deliv-ery use, with their emphasis on either en-capsulation or release. In order to achievetwo or more functions in one carrier system,different stimuli are normally required. Forexample, UV irradiation is applied to facil-itate cargo substance encapsulation, whereasexternal pH adjustment is used to adjustshell permeability and hence to triggerrelease;11 cargo substances are encapsu-lated first, and then pulsed light or near-infrared laser is used to rupture capsuleshells for release.12 Actually, rupture of thecapsule could be also wavelength-selectiveif capsule composition involves nanostruc-tures with different absorption spectra.13 Asthe essential part of light-responsive cap-sules, UV-responsive capsules whose func-tionalities can be accomplished by theexternal UV light make their contributions

* Address correspondence tog.sukhorukov@qmul.ac.uk.

Received for review June 12, 2013and accepted August 1, 2013.

Published online10.1021/nn4029772

ABSTRACT By introducing UV-sensitive chemical groups causing

different potential response as building blocks, fabricated LbL

capsules can be endowed with dual UV-responsive properties in

specific layers. One block is responsible for fast capsule sealing and

the other for longer term capsule swelling and rupture. Therefore,

the multifunction of these capsules could be activated selectively

when exposed to external UV light with suitable wavelengths. In this

work, dual-functional complex microcapsules (PDADMAC/PAZO-

)4-(DAR/Nafion)2 containing both diazonium and aozbenzene groups were proposed as clear examples to realize a time-dependent UV response for

successive encapsulation and release. Upon exposure to UV light, the DAR/Nafion layers underwent a rapid in situ cross-linking and hence to seal the

capsule shells through diazonium-related photolysis. Then further gradual shell swelling was followed by realignment of azobenzene molecules in

PDADMAC/PAZO layers. Fluorescent polymers were consequently studied as cargo substances. Results indicated that continuous UV light triggered rapid

cargo encapsulation over minutes time scale and gradual release with continuous irradiation over hours.

KEYWORDS: diazonium . azobenzene . microcapsule . encapsulation . release

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to the applications in surface science and environmen-tal areas. With regard to the practical use, sometimesthe continuous UV light (e.g., sunlight) would be theonly one stimulus available to functionalize thesesystems. Therefore, the great importance and chal-lenge here would be the development of multifunc-tional capsule systems, which would integrate multi-functionalities such as both encapsulation and releasein one system, simply triggered by only one externalstimulus. However, to the best of our knowledge, nosuch UV-responsive LbL capsules have been devel-oped yet for possible applications.Considering the stepwise capsule fabrication proce-

dure, it is possible to achieve the goal by introducingfunctional UV-responsive chemical groups (components)in one capsule system whose reactivity leads to vari-able response. Strategically, the encapsulation andfurther release could be activated through successiveUV-triggered chemical transitions of these functionalgroups. Ideally, with proper control over the balance ofcapsule shell structures, UV light with continuouswavelengths (containing the effective working wave-lengths for corresponding functional groups) couldpush forward a UV-exposure-dependent progress ofcapsule shell sealing and breakage (swelling and/ordisruption). On one hand, in order to realize encapsula-tion, a fast and efficient sealing effect, which decreasesshell permeability by making the shell dense, eliminat-ing voids, and expelling water from multilayers, istherefore favored. The building blocks containing UV-sensitive diazonium groups, such as diazo-resin (DAR),could be used as optimal candidates to seal capsuleshells by converting the paired ionic groups intocovalent bonds rapidly.14 On the other hand, releaseof the encapsulated substance can be accomplishedby continued UV exposure to induce shell breakage, ineither instant or sustainedway. Typically, a slow releaseover an extended period can be achieved by incorpor-ating the robust azobenzene molecules into multi-layers. These molecules could undergo a UV-triggeredmolecule realignment in the form of end-to-end(J-styled) or face-to-face (H-styled) aggregates, result-ing in phase separation in microdomains and furtherdestruction of shell integrity.15 Therefore, cargo sub-stance encapsulation and further release triggered by asingle UV source can be integrated in one complexcapsule system, benefiting from opposite UV responseof diazonium and azobenzene groups differing by timeinterval.In this work, we are aiming to fabricate a unique

dual-functional complex microcapsule system by in-troducing two different UV-sensitive chemical groupsin multilayer shells. In order to achieve the desired fastshell sealing and gradual breakage triggered by UVlight, capsules with PDADMAC/PAZO and DAR/Nafionlayers were studied as typical examples here. Undera given UV irradiation condition, the fast shell sealing

effect could be completed by in situ cross-linking ofDAR/Nafionmultilayers througha short-termUV-inducedphotolysis (e.g., minutes). Further gradual breakagewould be accomplished by realignment of azobenzenemolecules in PDADMAC/PAZOmultilayers triggered bylong-term UV irradiation (e.g., hours). Promisingly,these fabricated capsules would be able to encapsu-late cargo substances due to the charge eliminationand resulting hydrophobic structures and then realizecontrolled release through shell breakage originatingfrom local azobenzene molecular self-reorganization.The morphology and stability changes induced by UVirradiation of these complex capsules were studied.Moreover, UV-triggered cargo substance encapsulationand further release were also investigated.

RESULTS AND DISCUSSION

Gradual Capsule Breakage Based on Realignment of Azoben-zene Molecules. Exposure of (PDADMAC/PAZO)4 micro-capsules to UV light led to a gradual capsule swellingand further disruption process, as shown in Figure 1.Before UV irradiation, these capsules exhibit flat andintact images under SEM observation. After 10 min ofUV irradiation, capsules were observed as swollen oneswith an average size increase from 5.12 to ∼7 μm(Figure 1b). With the improved size increase effect, theintegrity of shell formations was destroyed, as most ofthe capsules were found to be broken when the UVexposure duration reached 1 h (Figure 1c). Extendingthe UV irradiation to 2 h, no intact capsules could befound, with most of them torn into lamellar- andneedle-like formation (Figure 1d).

A typical feature of azobenzene molecules underUV exposure is their changes in dipole moment,although the azobenzene molecular conformationchange in-plane is themost well-known phenomenon.Depending on themutual orientation of the interacteddipole moments between the counterpart molecules,azobenzenee moieties are conducted to form end-to-end or plane-to-plane aggregates, also known as J- orH-aggregates, respectively.16 Such styled aggregatescan easily be monitored by UV�vis spectroscopicmeasurements technically, exhibiting as maximumabsorption peak shift, red shift for J-aggregates andblue shift for H-aggregates. As shown in Figure 2a, aspectral change was visualized as a time-dependentshift toward the longer wavelength region. Generally,exposing the (PDADMAC/PAZO)4 microcapsule sus-pension to the UV source led to a red shift by 17 nmand subsequently receding to a constant maximumabsorption peak located at 383 nm. Compared to thelimited decrease of maximum absorbance, which re-presented the possible photoisomerization of azoben-zene, the red shift was found to be much pronounced.Therefore, it was believed that the J-aggregate forma-tions of azobenzene were favored in the PDADMAC/PAZO capsule system.

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In this work, UV irradiationwas able to cause irrever-sible disruption of PDADMAC/PAZO microcapsules.However, another polyelectrolyte microcapsule sys-tem (PAH/PAZO)3PAH/PVS composed of PAZO, poly-(allylamine hydrochloride) (PAH), and poly(vinylsulfo-nate) (PVS) demonstrated an opposite phenomenonafter UV irradiation, where the capsules were shrunksignificantly accompanied by apparent shell perme-ability decrease, as reported by Bédard and co-workers.8

As proposed in their work, the UV-induced trans tocis isomerization of the azobenzene molecules was

attributed to the main reason that caused capsuleshrinkage. Thus one would raise the hypothesis thatthe different UV-induced capsulemorphology changescould be originated from different chemical transitionsoccurring in these multilayers, where structural con-formation change in-plane or molecular realignmentlocally plays the dominant role.

Whether to undergo a structural conformationchange or a molecular realignment mainly dependson the interactions of azobenzene derivatives and theirpolymeric counterparts. When a polymer PAZO bearing

Figure 1. SEM images of (PDADMAC/PAZO)4 capsules after UV irradiation of 0 (a), 10 min (b), 60 min (c), and 120 min (d).

Figure 2. UV�vis spectra of (PDADMAC/PAZO)4 microcapsule suspensions before and after UV irradiation (a), and schematicillustration of (PDADMAC/PAZO)4 microcapsule disruption induced by UV irradiation (b).

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azobenzene groups on its side chains is used as thepolyanion for microcapsule preparation, the interplayof PAZO with its polycation is very important. Polyca-tions with different chain flexibilities and charge dis-tributions show different influence on fabricatedmultilayers with PAZO. For example, when a linearpolyethylenimine (PEI) was used as the polyanion, ablue shift toward shorter wavelength in a UV�visspectroscopy was observed, and this tendency be-came predominant when increasing the number ofdeposited PEI/PAZO layers.17 Combination of PAZOwith relatively flexible PAH and PVS polymers led toconformational alteration of azobenzene moleculeaccompanied by a length reduction of this group from9.0 to 5.5 Å.8 Contrarily, when the polymer PDADMACbearing five-membered heterocycles and ionic chargesalong the backbone structure was used as the polyca-tion, deposition of PDADMAC/PAZOwas not as regularas that of other paired polyelectrolytes; sometimes theinterdigitation of PDADMAC and PAZO polymer chainswas preferred instead of proper deposition in thecorresponding multilayer films.17 Such irregular poly-electrolyte deposition normally led to closely packedformation of 60 nm aggregates, exhibiting as patchystructures of the films.17 Formed aggregates demon-strated steric hindrance to restrict the photoisomeriza-tion of azobenzene molecules and consequentlyfacilitated the molecular realignment.18,19

UV-induced aggregates have been used as usefultriggers to modify fabricated thin films; typical applica-tions are electrical conductivity switching20 and liquidcrystal alignment.21 In the case of shell-like formations,it was reported that the aggregates or assembledaggregates of azobenzene derivatives induced by UVirradiation can lead to “catastrophic” destruction of theshell integrity, which has been developed as a strategyto build up photosensitive liposomes22 and polymer-somes23 for drug delivery use. Here, the gradual cap-sule breakage process in our (PDADMC/PAZO)4 cap-sule system could be explained as the influence ofthese end-to-end J-aggregates as in-line effected byPDADMAC, as shown in Figure 2b. PAZO polymers withanisotropic azobenzene moieties were deposited onspherical templates together with PDADMAC to fabri-cate microcapsules (Figure 2b, stage 1). When exposedto UV light, the azobenzene molecular motion locallywas found predominantly in a PDADMAC/PAZO multi-layer system, where the rigid PDADAMC chains re-stricted the azobenzene molecular trans to cis con-formation change and favored formations of J-styledaggregates (Figure 2b, stage 2). Assembled aggregatesgenerated steady formations as mosaics or lattices ofthe small “unit” aggregateswith a surface area of∼20Åvia strong noncovalent aromatic�aromatic interac-tions in the presence of water (Figure 2b, stage 3).24,25

The numerous formations of such mosaic aggregatesmade the thin spherical shells lose their flexibility. Once

the capsules became not flexible enough to keep thespherical shell structures, their integrity was conse-quently lost; then these capsules gradually started tobe spilt locally (Figure 2b, stage 4), demonstratingcapsule swelling phenomena and further disruption.Thus, at given composition of 4 PDADMAC/PAZObilayers, the destruction effect would be hours long.

Rapid Capsule Sealing Induced by Diazonium-Related Photo-lysis. DAR (Figure 4, inset) is a well-known UV-sensitivepolymer bearing diazonium groups on the side chains,which could be decomposed fast and readily whenexposed to UV light with a wavelength at ∼380 nm.Therefore, the UV irradiation here predominantly acti-vated the diazonium groups to form a phenyl cationand then to be substituted by nucleophilic groups,14

specifically the paired sulfonate groups of the poly-anion involved in LbL assembly. In this work, themultilayer capsules composed of DAR and Nafion wereprepared; then postpreparation UV treatment allowedan in situ chemical transition from weak ionic interac-tions to covalent bonding inmultilayers. Different fromthe (PDADMAC/PAZO)4, UV irradiation did not causeobvious morphology changes on the (DAR/Nafion)microcapsules; neither notable shell shrinkage norbreakage could be found, as shown in Figure 3. Asper the spectroscopic study shown in Figure 4a,b, after10 min of UV irradiation, the absorption peaks at 2222,2169, and 1580 cm�1, which represented the stretch-ing of �N2

þ26 and �CdC� in the phenyl group con-jugated with the diazonium group,27 disappearedcompletely. This could be explained as the fast decom-position of diazonium. However, due to the existenceof broad C�F2 stretching of Nafion,28 a new peak at1162 cm�1 representing the generation of sulfonatecoupled with the phenyl group26 was overlapped. ThisUV-induced photolysis occurred very rapidly, and itcould be completed within 1 min.14 As found in ourwork, 10 min of UV irradiation at 55 mW cm�2 wasadequate, extending UV treatment duration wouldlead to no remarkable chemical change. Unlike otherUV-induced chemical transitions withinmultilayer cap-sules, this DAR-related change required no polymerchain rearrangement29 or molecular conformationisomerization,8 thus no obvious capsule size changecould be found in this work and in similar systems.30

Introducing the diazonium groups into multilayersoffers a novel approach to seal the fabricated capsules,where the in situ cross-linking is triggered by UV light.This strategy provides the possibility to encapsulatecargo substances. An example has been reported byZhu and co-workers. In their work, the microcapsulescomposed of DAR/poly(styrene sulfonate) (PSS) wereable to encapsulate macromolecules with molecularweight from 9.5 to 186 kDa.30 However, one mustnotice, for the porous LbL assembled polyelectrolytecapsule shells, it is still a challenging task to realizesmall molecule encapsulation, especially for these

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Figure 4. Left panel: FTIR spectra of (DAR/Nafion)4 capsules before (a) and after 10 min of UV irradiation (b). Right panel:CLSM image of RhB encapsulation in irradiated (DAR/Nafion)4 microcapsules (c,d). Inset shows the underlying photolysis ofpaired charges.

Figure 3. SEM (top panels) and CLSM (bottom panels) images of (DAR/Nafion)4 microcapsules before (a,c) and after (b,d) UVirradiation.

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drugs, dyes, and other bioactive substances with mo-lecular weight below 1 kDa.31 To overcome this diffi-culty, Nafion, which consists of a perfluorinatedbackbone and contains sulfonic acid groups in shortside chains,32 is chosen for capsule fabrication togetherwith DAR. After UV treatment, elimination of pairedcharges and resulting hydrophobic shell structuresmade these irradiated capsules become “sealed” withless water carrying ability andminimal permeability. Asshown in Figure 4c,d, these sealed (DAR/Nafion)4capsules show good retention for the small moleculeRhB (Mw = 479). Thus, appropriate sealing could beachieved in minutes.

Combination of Both Capsule Sealing and Breakage. Asinspired by the completely opposite effect ofUV-responsive multilayers, but at variable timepoints, UV-triggered cargo substance encapsulation andrelease abilities can be integrated in one complexcapsule system. Ideally, with proper control over thebalance of reactive chemical groups (diazonium andazobenzene), UV lightwith continuouswavelength caninitiate a UV-exposure-dependent capsule sealing (whichis attributed to diazonium-related in situ cross-linking)and shell breakage (which is due to realignment oflocal azobenzene molecules). Practically, the balancecontrol could be achieved through adjustment of theratio of twomultilayer systems (DAR/Nafion and PDAD-MAC/PAZO), which might have different influence onbuilt capsules. Regarding the strengthened shell struc-tures of capsule systems containing Nafion after seal-ing, which required combination of freeze�thaw andsonication treatments to break them,33 the multilayerscontaining a small percentage of cross-linking sites(diazonium of DAR) would be preferred. In this work,complexmicrocapsules (PDADMAC/PAZO)4-(DAR/Nafion)2containing both azobenzene and diazonium groupswere studied as typical examples, to which four PDAD-MAC/PAZO and two DAR/Nafion layers were introduced.

As shown in Figure 5 (first row), the preparedmicrocapsules were flat with creases and folds underSEM observation. Without UV irradiation, these cap-sules showed uniform size distribution, with an aver-age diameter of 4.92( 0.32 μm. At high magnification(120k�), these capsules exhibited intact and relativelysmooth surfaces (Figure 5c). With the constant UVirradiation intensity (55 mW cm�2), complex capsulesexhibited a time-dependent swelling process afterexposure to UV light, as shown in Figure 5 and Figure 6.After the first 10min of UV irradiation (Figure 5, secondrow), no obvious size change could be found whencompared with the initial ones (Figure 5, first row), andthe capsules possessed an average diameter of 4.95(0.26 μm and the shell formations seemed intact; noobvious pore or crack could be observed. Therefore,the first 10 min of UV irradiation could be chosen asthe treatment to seal the outmost DAR/Nafion layers,which will be discussed later. When the UV irradiation

was extended to 20 min (Figure 5, third row), theaverage capsule size increased to 5.41( 0.61 μm. Afterexposure to UV for 30 min (Figure 5, fourth row), thecapsules swelled to 5.57 ( 0.43 μm in diameter, andobvious pores in the size of 30�40 nm emerged on theshells. Further extending the UV irradiation duration,most of the capsules continued to increase their sizeand the numbers of small pores on their shells. After 3 hof UV irradiation (Figure 5, last row), almost all of thecapsules were swollen, and some of them possesseda size of >8 μm; as a consequence of continuousUV irradiation, the capsule surfaces appeared muchrougher due numerous pores with their size up to100 nm formed on the shells. After that, further UVirradiation led to no visible effect on the capsule shellmorphology and stability.

For the polyelectrolyte solutions, maximumabsorp-tion peakswere found at 358 and 372 nm for PAZO andDAR, respectively. In theory, due to the analogous UVabsorption curves and neighboring maximum UV ab-sorption peaks, the absorption of the complex capsulesshould exhibit as a compromise of azobenzene anddiazonium groups. However, the prepared multilayercapsules showed an absorption shift slightly towardlonger wavelength region when the two polymerswere assembled with the other two polyelectrolytes(PDADMAC and Nafion). As shown in Figure 7, beforeUV irradiation, a strong absorption peak centered at372 nm was found, which was contributed by theabsorption of both azobenzene (π�π* transition at∼360 nm) and diazonium (π�π* transition at∼380 nm),and the weak peak detected at 270 nm was attributedto their parallel or concomitant absorption.17,26 Withexposure to UV irradiation, a fast absorbance decreasewas observed. This result was attributed to photolysisof diazonium groups and possible photoisomerizationreaction of azobenzenemoieties if there was any. After10 min of irradiation, the absorption intensity de-creased to 50% of the initial absorbance. Consideringthe SEM image of complex capsules after 10 min of UVirradiation (Figure 5, second row), potential chemicalchanges occurring during this period would not causecapsule swelling.

Practically, the DAR-related photolysis took placevery fast, and a previous report demonstrated a com-pleted cross-linking effect within 50 swith treatment ofa 80 W mercury lamp at a distance of 13 cm.14 For the(DAR/Nafion)4 capsules, we have already found that10min of UV irradiationwas sufficient enough to decom-pose all the diazonium groups and to eliminatethe maximum absorption with a UV intensity of50 mW cm�2. However, a corresponding peak did notdisappear after 3 h of UV irradiation in this work. Thisresult should be attributed to the existence of PDAD-MAC/PAZO layers. As suggested, the photoisomeriza-tion of the azobenzene molecule could not becompleted in this system due to the steric hindrances

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against trans to cis conformation changes as well aspossible hindrances of azobenzene aggregates, whosepossible mechanism of formation has been discussed

already.17�19 Therefore, the remaining absorption wasassigned to that of PDADMAC/PAZO layers, whichcannot disappear in 3 h of UV irradiation.

Figure 5. SEM images of complex microcapsules before (first row) and after UV irradiation of 10 min (second row), 20 min(third row), 30 min (fourth row), 1 h (fifth row), 2 h (sixth row), and 3 h (last row) at different magnifications.

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As mentioned above, the UV irradiation wouldfacilitate realignment of azobenzene molecules in thePDADMAC/PAZO system, resulting in formation ofJ-aggregates. The generation of such aggregates wasalso detected in the complex capsule system. As shownin Figure 7, a time-dependent shift of maximum ab-sorption toward longer wavelength was found. Briefly,after 10min of UV irradiation, themaximumabsorptionwas shifted by 4 nm and located at 376 nm. Furtherexposing to UV light led to a slow red shift. When theUV irradiation reached 3 h, a total red shift by 8 nmwasvisible at 380 nm.

The FTIR spectra of complex capsules before andafter UV irradiation are shown in Figure 8 in order todemonstrate possible chemical changes in detail. After10 min of UV irradiation, the peaks at 2222 and2162 cm�1 representing stretching of �N2

þ� disap-peared completely. With the disappearance of thepeak at 1577 cm�1 (�CdC� in the phenyl groupconjugated with the diazonium group), a new peakrepresenting a normal absorption of the phenyl group,which was overlapped by the signal of the diazoniumgroup before, was observed at 1590 cm�1. All these

changes should be explained as the UV-induceddecomposition of diazonium groups in the multi-layers.26,27 Meanwhile, another peak at 1111 cm�1

corresponding to the N�O stretching vibrationalmode of complexes formed within DAR and the ad-jacent polyanion also disappeared. Considering theexistence of both Nafion and PAZO polymers surround-ing DAR, referring to the shell structure (PDADMAC/PAZO)4-(DAR/Nafion)2, this peak should be attributedto N�O stretching of complexes of diazonium andsulfonate groups (in Nafion) (�N2

þ fOSO2�)34 and

also the complexes of diazonium and carboxylategroups (in PAZO) (�N2

þ fOCO�).35 The disappear-ance of this peak also confirmed decomposition ofdiazonium groups under UV irradiation.

Possible photoisomerization of azobenzene mol-ecules changing from trans to cis could be easilymonitored by FTIR, demonstrated by the disappear-ance of a trans-NdN stretching vibration at 1380�1400 cm�1 and generation of cis-NdN stretching at∼1460 cm�1 as reported in related research.36,37 How-ever, when the UV irradiation duration reached 3 h, noobvious changes due to azobenzene molecular con-formation change in-plane could be observed in thiswork. Combining the found red shift effect in UV�visspectroscopy (Figure 7), the J-aggregation was hencebelieved to be the predominant transition in thisduration, although the degree of aggregation wasfound not to be as significant as that in the purePDADMAC/PAZO system (Figure 2a).

Generally, the UV-induced chemical changes inthese complex capsule systems are superimposed,involving in situ covalent bonding between paireddiazonium and sulfonate groups (Scheme 1a,b) andJ-styled realignment of azobenzene molecules whichare mostly influenced by the interplay of PDADMACpolymers (Scheme 1c,d). One should notice, as pre-sented in the (PDADMAC/PAZO)4-(DAR/Nafion)2 struc-ture, the diazoniumof DAR not only interactedwith thesulfonate groups of Nafion but also paired with

Figure 6. Size changes of complex capsules after UV irra-diation. Capsule diameters and distributions were ex-pressed as mean ( SD of at least 30 capsules per sampleof random measurement of SEM images.

Figure 7. UV�vis spectra of complex microcapsules uponexposure to UV light.

Figure 8. FTIR spectra of complex capsules before (a) andafter UV irradiation for 10 min (b) and 3 h (c).

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carboxylate groups of PAZO. As a consequence, theinteraction between the PDADMAC and PAZO wassomewhat weakened, while the effect of DAR-relatedsealing was strengthened by the appearance of cross-linking between PAZO and adjacent DAR layers. There-fore, the UV-induced red shift in complex capsules wasnot as significant as that observed in the capsules com-posed of only PDADMAC/PAZO layers (Figure 2a). Inreturn, in the complex capsule system of (PDADMAC/PAZO)4-(Nafion/DAR)2, the potential azobenzene mo-lecular motion as J-aggregates occurred in the PDAD-MAC/PAZO layers was not powerful enough to causeobvious phase separation or patch-like structures inmacroscale. Therefore, the intrinsic chemical transitionhere primarily led to swollen microcapsules (sizechange from 5 to 8 μm) and generation of nanoscalepores on the shell. Differently, similar UV-inducedazobenzene realignment usually tore the shell-like

formations into pieces, demonstrating lamellar- orcrystal-like formations.22 However, in this work, onlyvery few broken capsules and lamellar-like formations(symbol Δ) can be found occasionally after 3 h of UVirradiation, as shown in Figure 9.

Complex Capsules for Encapsulation and Release Triggered byContinuous UV Light. Practically, 10 min of UV irradiationwith an intensity of 55 mW cm�2 can accomplish theDAR-related photolysis reaction, which could convertthe electrostatic interacted charges into covalent che-mical bonds, as discussed above. This cross-linkingwithin paired ionic groups provided adequate capabil-ity to seal the shells and therefore to entrap the cargosubstance inside.30 Here, efforts were devoted to en-capsulate molecules in the complex capsules, in whichthe DAR/Nafion multilayers would serve as potentialsealing layers to achieve the goal. As the SEM imagespresented in Figure 5, UV irradiation had a significant

Figure 9. SEM images of a broken complexmicrocapsule (a) and lamellar-like formations (b) after UV irradiation at 3 h. Imageb shows the magnified area of interest; the symbol Δ shows the lamellar-like formations.

Scheme 1. Schematic illustration of UV-induced complex capsule shell sealing and further swelling.

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effect on the morphology changes of complex cap-sules, exhibiting a time-dependent swelling progress.In particular, no obvious capsule size increase or shellporosity change can be observed after the first 10 minof UV irradiation, thus this UV treatment duration waschosen here as a typical working duration for capsulesealing. On the other hand, considering the long UVtreatment duration that would be applied to triggerrelease of encapsulated substances, fluorescent poly-mers with high photostability should be used here.Strategically, Alexa Fluor-labeled dextran (AF488-dex-tran, 10 kDa) was studied because it has been reportedthat the sulfonic acid substituents of Alexa Fluor couldincrease water solubility and inhibit dye�dye interac-tions, which made the Alexa Fluor dyes brighter andmore stable than common dyes (e.g., fluorescein andrhodamine), reducing quenching and bleaching.38

Moreover, AF488-dextran has been used in otherresearch works for fluorescent visualization and en-capsulation study.8,39

After the first 10 min of UV irradiation, the complexcapsules were sealed, and the AF488-dextran wastherefore retained inside, visualizing as bright fluores-cent image in capsule cavities; very strong fluorescentsignal intensity (more than 250 units) was detected(Figure 10a). Further UV irradiation caused a gradualcapsule swelling progress (Figure 5), resulting in therelease of encapsulated AF488-dextran. After 7 h of UVirradiation, only hollow capsules with limited fluores-cent signal can be observed (Figure 10b). Meanwhile,some of the swollen capsules were found to be col-lapsed in water (pointed out by the arrow). Contrarily,the capsules without the PDADMAC/PAZO layers,(DAR/Nafion)4 to be specific, demonstrated a constant

fluorescent signal intensity before and after 7 h of UVirradiation. This result was attributed to the UV-in-duced sealing effect of the DAR/Nafion layers, whichcan predominantly seal the multilayer shells and notbreak complex capsules at relevant time points.

The released mass of AF488-dextran from sealedcomplex capsules was then investigated, released eitherby UV irradiation or by diffusion. In the meantime, anAF488-dextran solution was also irradiated as thecontrol group, in order tomonitor possible UV-inducedphotobleaching effect under the same UV treatmentcondition. Its concentration was adjusted to be20 μg mL�1, or 8000 ng of AF488-dextran in 400 μLvolume to be specific. This amount roughly matchedthe mass of encapsulated AF488-dextran in each por-tion of complex capsule sample, as determined fromthe results of preliminary experiments.

The mass of the encapsulated AF488-dextran wasdetermined to be 7.2 mkg inside 6.7 � 106 capsules inthis work, which meant that 1.1 pg of AF488-dextranwas entrapped in one capsule. In the 7 h of UVtreatment duration, the control group (irradiated pureAF488-dextran solution) showed good stability againstUV light. After 7 h of UV treatment, there was still7.8 mkg of AF488-dextran detected in each portion,exhibiting a roughly constant mass of AF488-dextranat each set experimental time point (Figure 11, inset).Therefore, it could be assumed that the influence of UVirradiation on dye photostability of AF488-dextransolution here (20 μg mL�1) was negligible, and thedata of UV-triggered AF488-dextran release could bereliable in this duration.

Generally, with the increase of UV irradiation time,the detected AF488-dextran tended to increase, asshown in Figure 11. For each sample containing6.7� 106 complex capsules, after 10 min of UV irradia-tion, 0.87 mkg of released fluorescent polymers wasfound. When UV irradiation was extended to 30 min,1.24 mkg was then detected. When the UV irradia-tion duration reached 4 h, more than 50% of AF488-dextren (3.9 mkg) was released from the capsules.

Figure 10. CLSM images of AF488-dextran encapsulation incomplex (top panel) and (DAR/Nafion)4 (bottom panel)capsules right after shell sealing (a,c) and after 7 h ofadditional UV irradiation (b,d). The line scan insets showedrelative fluorescent intensity in capsules; the arrows repre-sent collapsed capsules in water.

Figure 11. Mass of UV-triggered AF488-dextran releasefrom complex capsules. The inset shows the detected massof AF488-dextran of the control group after UV irradiation.

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After 7 h, 5.0 mkg of the AF488-dextran was found,which demonstrated a UV-triggered release efficiencyof 70.5%. It should be noted here that not allthe encapsulated AF488-dextran was released (asdetected) even after 7 h of UV irradiation. Besidespossible photobleaching effect, this result could beexplained by the adsorption of the fluorescent poly-mers on capsule shells, as some green “rings” can beseen under CLSM observation (Figure 10b). On thecontrary, for the capsules kept in the dark, the fluor-escent polymer release was found to be quite slowwhen compared to that of the irradiated ones. At thebeginning, 0.67 mkg of AF488-dextran was expelledfrom the diffusive shells. With an increase of incubationtime, the release by diffusion through the multilayersshowed a continuously increasing tendency. However,there was only 2.2 mkg of AF488-dextran detected after7hof incubation, 31%of the initial encapsulatedamount.

It is worth mentioning that the UV-triggered com-plex capsule successive sealing and disruption is basedon UV absorption in the first instance. Generally speak-ing, we have two effects such as absorption of PAZOwhich occurs in the range of 330�450 nm and inducedreactivity of the DAR component at 380 nm at max-imum. Thus, these intervals that almost overlappedrestrict the separation of both effects by tuning wave-length. Fine narrowing wavelength for UV irradiation,for instance, by using UV lasers instead of a lamp,would have a different effect on DAR and PAZOcomponents, and hence, the two adverse effects inmultilayers could not be interfered with timing. On theother hand, decrease of the UV intensity might affectthe timeline of the process. In the present work, the UVirradiation was set at an intensity of 55 mW cm�2 tokeep all conditions the same throughout the experi-ments. We did the experiment at half intensity of 27mW cm�2, and the release rate of cargo from PAZO/PDADMAC capsules was lowered, reflecting retentionof PAZO block rearrangements in the multilayers. Onewould suggest that at lower intensities the sealingprocessmight get longer, as well. However, the ruptureeffect may overlap with the sealing, considering thatthe capsules are not absolutely identical in population.Overall result might be the smearing effect of bothshell sealing and disruption in the course of time.

Apart from the intensity and wavelength of UVirradiation, the multilayer shell architecture plays a

key role in UV-induced capsule mechanical change.Likewise, the same UV irradiation time could havedifferent effects on capsule with varied layer constitu-ents. Speculatively, one would suggest that there mustbe a minimum number of each component layerrequired in order to realize the UV-stimulated effectsuch as either sealing or rupture. It is hard to anticipatewhat would happen if more functional polyelectrolytelayers are assembled. This might be a subject for futurestudy. At present, careful considerations should begiven to design of the dual-functional complex capsulesystems, where the influencing factors (e.g., UV dosage,capsule shell architectures, ratio of functional groups)would have subtle effects on the capsules' potential UVresponse.

CONCLUSIONS

UV triggered dual-functional complex microcap-sules were fabricated in this work for the purpose ofintegrating both encapsulation and release in onecapsule system. Two multilayer systems effecting dif-ferently, PDADMAC/PAZO and DAR/Nafion, were in-troduced to build up complex capsules by using LbLassembly technique. Exposure these capsules to10 min of UV irradiation (at ∼380 nm) led to diazonium-related photolysis within DAR and adjacent Nafion orPAZO layers. This short time UV irradiation eliminatedthe paired charges and facilitated cargo substanceencapsulation due to densification of multilayer shells.The successful encapsulation of fluorescent polymerssuch as AF488-dextran has been demonstrated. Later,exposure of these capsules to longer UV treatmentduration (at ∼360 nm) caused an irreversible shellswelling progress, which was activated by the pre-ferred J-aggregations of azobenzene molecules inPDADMAC/PAZO multilayers, providing a way to re-lease the encapsulated substances. Generally, UV-trig-gered release of AF488-dextran was investigated as atypical example. Promisingly, these complex capsulescontaining two different functional multilayers wouldbe useful microcontainers for various applications, forwhich their entrapment, retention, and controlledrelease could easily be trigged by continuous UV light.Hopefully, our strategy to fabricate such dual-functioncomplex capsules, which could be activated by singleexternal stimulus, would inspire the engineering ofmultifunction vesicles for potential applications.

MATERIALS AND METHODS

Materials. Alexa Fluor 488-labeled dextran (AF488-dextran,10 kDa) was purchased from Invitrogen. Silicon dioxide micro-particles (4.99 ( 0.22 μm) were purchased from microparticlesGmbH (Germany). Nafion perfluorinated resin (10 wt % in H2O),poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO,Mw∼ 100 kDa), poly(diallyl-dimethylammonium chloride) (PDADMAC, Mw 200�350 kDa,

20 wt % in H2O), paraformaldehyde, diphenylamine-4-diazo-nium salt (Variamine Blue RT Salt), Rhodamine B (RhB), and otherchemicals were purchased from Sigma-Aldrich. All the chemi-cals were used as received without further purification.

Methods. Capsule Preparation. Diazo-resin (DAR) was synthe-sized through a polycondensation reaction of diphenylamine-

4-diazonium salt with paraformaldehyde, following an electrophi-

licmechanism,14 andusedaspositively chargedpolyelectrolyte for

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capsule fabrication. Polyelectrolytemultilayerswere assembled onthe SiO2 particles by using the LbL assembly technique,40 startingwith positively charged polyelectrolytes (PDADMAC or DAR). Inorder to ensure a better attachment of first polymer deposition,these SiO2 templates were first treatedwith amixture of 25%NH3/30% H2O2/H2O (1:1:5) for 15 min at 75 �C prior to the assembly.41

To avoid aggregation, polymer-coatedparticleswere sonicated for10 s after each wash step. After the polymer deposition steps,the sacrificial templates were removed by treatment with a0.2 M NH4F and HF buffer solution at pH = 4.5.42 Microcap-sules (PDADMAC/PAZO)4, (DAR/Nafion)4, and complex micro-capsules (PDADMAC/PAZO)4-(DAR/Nafion)2 were obtainedafter several wash steps.

UV-Induced Encapsulation and Release. To detect the feasi-bility of encapsulation in complex capsules, fluorescent smallmolecule (RhB) and polymer (AF488-dextran) were used asmodel cargo substances. Briefly, fabricated microcapsules wereredispersed in these fluorescent solutions (300 μg mL�1) for 2 hwith shaking. Then the mixtures were treated with UV irradia-tion for 10min, washed several times to remove free fluorescentsubstances in water, and observed with a Leica TS confocalscanning system (Leica, Germany) equipped with a 63�/1.4 oilimmersion objective.

For quantification of released fluorescent polymer (AF488-Dextran, 10 kDa) from complex microcapsules, 1 mL of capsulestock suspension (containing 1 � 108 capsules) was resus-pended in 2.0 mL of AF488-dextran solution (300 μg mL�1)for 2 h with shaking. The capsule�dye mixtures were exposedto UV light directly for 10 min. After shell sealing, capsules werecollected and washed several times with water to remove freepolymers. The resulting suspension was then split into twoportions (diluted to 3mL for each); one portion was treatedwithadditional UV irradiation up to 7 h, and the other portion waskept in the dark. After a set UV experimental time point, 400 μLof capsule�dye mixture (containing 6.7 � 106 capsules) with/without additional UV treatmentwas taken out and centrifuged,the supernatant was carefully collected, and the precipitate wasadded with equal volume of pure water. The fluorescenceintensity of each sample (in supernatant or in precipitate) wasdeterminedwith a fluorescence spectrometer (Perkin-Elmer LS 55)and normalized with the standard fluorescent polymer solutionswith known concentrations.

Instrument and Measurement. A UV lamp (OmniCure 2000,Lumen Dynamics Group Inc.) with effective working wave-length ranging from 320 to 500 nm was used to irradiate thesamples. Continuously stirred capsule suspensions were ex-posed to the UV source directly. The UV intensity used in thiswork was detected to be approximately 55 mW cm�2.

UV�visible (UV�vis) absorption spectra of polyeletrolytesand capsule suspensions were determined by using a spectro-photometer (LAMBDA 950, Perkin-Elmer). Aqueous solutionmeasurements were performed using quartz spectrophot-ometer cuvettes (S10C, Sigma).

Capsule morphologies were provided by scanning electronmicroscopy (SEM) (FEI Inspect-F). Diluted capsule suspensionwas dropped on silicon wafer, air-dried, and coated with gold.SEM observation was carried out using an accelerating voltageof 10 kV, a spot size of 3.5, and a working distance of approxi-mately 10 mm.

An infrared spectroscopy (FTIR spectrometer 100, Perkin-Elmer)was applied to detect the FTIR spectra of vacuum-dried capsulesamples. All the data were collected at a spectral resolution of4 cm�1.

Confocal laser scanning microscopy (CLSM) images werecaptured with a Leica TS confocal scanning system (Leica,Germany) equipped with a 63�/1.4 oil immersion objective.

Conflict of Interest: The authors declare no competingfinancial interest.

Acknowledgment. The authors thank Dr. AntonM. Pavlov ofthe School of Engineering and Materials Science (QMUL) for hishelp with the dye release study. The work was supported in partby L'Oreal (Paris, France). Dr. Andrew Greaves (L'Oreal) isthanked for stimulating discussions. Q.Y. thanks the SEMSCollege DTA Studentship (SEMS, QMUL) for her Ph.D. study.

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