DOI: 10.1021/la104232r 1275Langmuir 2011, 27(4), 1275–1280 Published on Web 12/03/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Modular Assembly of Layer-by-Layer Capsules with Tailored

Degradation Profiles†

Christopher J. Ochs, Georgina K. Such, and Frank Caruso*

Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville,Victoria 3010, Australia

Received October 21, 2010. Revised Manuscript Received November 8, 2010

Herein we report the preparation of layer-by-layer (LbL) assembled, biodegradable, covalently stabilized capsuleswith tunable degradation properties. Poly(L-glutamic acid) modified with alkyne moieties (PGAAlk) was alternatelyassembled with poly(N-vinyl pyrrolidone) (PVPON) on silica particles via hydrogen-bonding. The films were cross-linked with a bis-azide linker, followed by removal of the sacrificial template and PVPON at physiological pH throughhydrogen bond disruption, yielding one-component PGAAlk capsules. To control the kinetics and location of capsuledegradation, a number of approaches were investigated. First, a degradable bis-azide cross-linker was incorporated intothe inherently enzymatically degradable capsules. Second, we assembled low-fouling capsules composed of nondegrad-able poly(N-vinyl pyrrolidone-ran-propargyl acrylate) (PVPONAlk) via hydrogen bonding with poly(methacrylic acid)(PMA) and combined this with the aforementioned system (PGAAlk/PVPON) to produce stratified hybrid capsules.The degradation profiles of these stratified capsules can be closely controlled by the number as well as the position ofnondegradable barrier layers in the systems. The facile tailoring of the degradation kinetics makes this stratified LbLapproach promising for the design of tailored drug-delivery vehicles.

Introduction

The development of functional and engineered therapeuticcarrier systems has received considerable and widespread interestover the past decade. A range of polymeric carriers have beendeveloped for delivery applications, including polymersomes,micelles, and polymer capsules.1-6 In particular, hollow polymercapsules prepared via layer-by-layer (LbL) assembly are ofinterest because they can be designed with a range of properties,such as enzymatic degradability,7-9 low-fouling surface pro-perties,10,11 and antibody functionalization for targeted drug

delivery.12-15 LbL capsules have been assembled from a varietyof polymers based on electrostatic interactions,16,17 hydrogenbonding,18,19 and click chemistry.20 Previous work has demon-strated that a range of properties can be engineered into LbLcapsules to achieve cargo release in response to external triggers,such as optical or magnetic stimulation,8,21-23 changes inpH,20,24-27 or (enzymatic) degradation.28-30

A common limitation of carrier systems is the initial burstrelease of the encapsulated cargo and the resulting rapid depletionof the drug reservoir. The ability to release multiple drugs atdefined rates and in programmable and predetermined intervalsremains an ongoing challenge for delivery systems. Controlled

† Part of the Supramolecular Chemistry at Interfaces special issue.*Corresponding author. E-mail: fcaruso@unimelb.edu.au.(1) Becker, A. L.; Johnston, A. P. R.; Caruso, F. Small 2010, 6, 1836–1852.(2) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. Rev. 2001, 53,

283–318.(3) Petros, R. A.; DeSimone, J. M. Nat. Rev. Drug Discovery 2010, 9, 615–627.(4) Ariga, K.; Hill, J. P.; Lee, M. V.; Vinu, A.; Charvet, R.; Acharya, S. Sci.

Technol. Adv. Mater. 2008, 9, 96.(5) Ariga, K.; Ji, Q. M.; Hill, J. P. Enzyme-Encapsulated Layer-by-Layer

Assemblies: Current Status and Challenges toward Ultimate Nanodevices. InModern Techniques for Nano- and Microreactors/-Reactions; Caruso, F., Ed.;Springer-Verlag: Berlin, 2010; Vol. 229, pp 51-87.(6) Stadler, B.; Price, A. D.; Chandrawati, R.; Hosta-Rigau, L.; Zelikin, A. N.;

Caruso, F. Nanoscale 2009, 1, 68–73.(7) Jung, J.; Lee, I. H.; Lee, E.; Park, J.; Jon, S. Biomacromolecules 2007, 8,

3401–3407.(8) Angelatos, A. S.; Radt, B.; Caruso, F. J. Phys. Chem. B 2005, 109, 3071–

3076.(9) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006, 7, 27–30.(10) Wattendorf, U.; Kreft, O.; Textor, M.; Sukhorukov, G. B.; Merkle, H. P.

Biomacromolecules 2008, 9, 100–108.(11) Heuberger, R.; Sukhorukov, G.; Voros, J.; Textor, M.; Moehwald, H.Adv.

Funct. Mater. 2005, 15, 357–366.(12) Cortez, C.; Tomaskovic-Crook, E.; Johnston, A. P. R.; Radt, B.; Cody,

S. H.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. Adv. Mater. 2006, 18,1998–2003.(13) Zhou, J.; Romero, G.; Rojas, E.; Moya, S.; Ma, L.; Gao, C. Y.Macromol.

Chem. Phys. 2010, 211, 404–411.(14) Kamphuis, M. M. J.; Johnston, A. P. R.; Such, G. K.; Dam, H. H.; Evans,

R. A.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. J. Am. Chem. Soc. 2010,132, 15881–15883.(15) Ariga, K.; Hill, J. P.; Endo, H. Int. J. Mol. Sci. 2007, 8, 864–883.

(16) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; M€ohwald, H.Angew. Chem. Int. Ed. 1998, 37, 2201–2205.

(17) De Geest, B. G.; Sanders, N. N.; Sukhorukov, G. B.; Demeester, J.;De Smedt, S. C. Chem. Soc. Rev. 2007, 36, 636–639.

(18) Fu, Y.; Bai, S. L.; Cui, S. X.; Qiu, D. L.; Wang, Z. Q.; Zhang, X.Macromolecules 2002, 35, 9451–9458.

(19) Kozlovskaya, V.; Kharlampieva, E.; Mansfield, M. L.; Sukhishvili, S. A.Chem. Mater. 2006, 18, 328–336.

(20) Such, G. K.; Tjipto, E.; Postma, A.; Johnston, A. P. R.; Caruso, F. NanoLett. 2007, 7, 1706–1710.

(21) Katagiri, K.; Nakamura, M.; Koumoto, K. ACS Appl. Mater. Interfaces2010, 2, 768–773.

(22) Bedard, M. F.; Sadasivan, S.; Sukhorukov, G. B.; Skirtach, A. J. Mater.Chem. 2009, 19, 2226–2233.

(23) Schneider, G. F.; Subr, V.; Ulbrich, K.; Decher, G.Nano Lett. 2009, 9, 636–642.

(24) Ochs, C. J.; Such, G. K.; Stadler, B.; Caruso, F. Biomacromolecules 2008, 9,3389–3396.

(25) Dejugnat, C.; Sukhorukov, G. B. Langmuir 2004, 20, 7265–7269.(26) Mauser, T.; Dejugnat, C.; Sukhorukov, G. B. J. Phys. Chem. B 2006, 110,

20246–20253.(27) Biesheuvel, P. M.; Mauser, T.; Sukhorukov, G. B.; Moehwald, H. Macro-

molecules 2006, 39, 8480–8486.(28) Ochs, C. J.; Such, G. K.; Yan, Y.; van Koeverden, M. P.; Caruso, F. ACS

Nano 2010, 4, 1653–1663.(29) Borodina, T.; Markvicheva, E.; Kunizhev, S.; Moehwald, H.; Sukhorukov,

G. B.; Kreft, O. Macromol. Rapid Commun. 2007, 28, 1894–1899.(30) De Geest, B. G.; Vandenbroucke, R. E.; Guenther, A. M.; Sukhorukov,

G. B.; Hennink,W. E.; Sanders, N. N.; Demeester, J.; De Smedt, S. C.Adv.Mater.2006, 18, 1005–1009.

1276 DOI: 10.1021/la104232r Langmuir 2011, 27(4), 1275–1280

Article Ochs et al.

release has been achieved using particulate or multicompart-ment systems.31-34 Kim et al. observed controlled multidrugrelease from degradable micelle-polyelectrolyte hybrid films.35

Recent work on artificial cells utilizes capsule systems loadedwith liposomes36-38 or small reservoir capsules39 to controlenzymatic reactions within the construct. In other work, Volod-kin et al. achieved the temperature-triggered release of dyes andsilver ions from stabilized liposomes embedded in planar LbLfilms.40-42 However, these approaches require separate synthesisand assembly of the reservoir compartments. Kreft et al. synthe-sized shell-in-shell microcapsules by the 2-fold coprecipitation ofdextran with calcium carbonate (CaCO3) and subsequent LbLshell formation.43,44 However, an external laser stimulus isrequired to trigger the spatially confined reactions, and theconditions necessary for the removal of CaCO3 limit this systemfor the encapsulation of pH-sensitive compounds. Related workby Borodina et al. reported the time-controlled release of DNAfrom self-disintegrating microcapsules by coloading degrad-ing agents into biodegradable polyelectrolyte shells.29 In thisapproach the shell disassembly relies on the encapsulated

degrading agent. Using a different approach, Garza et al. devel-oped a biodegradable multilayer system comprising polypeptide/polysaccharide reservoir layers separated by polyester barrierlayers.45,46 This system has so far been established only on planartemplates.

An effective approach to time-controlled release would com-prise the use of nondegradable barrier layers, hindering ordelaying degradation/release before the next drug reservoir ofdegradable material releases its contents. Thus, cascade reactionsand themultistep release of different drugs from one system couldbe achievedwith control of the release time and rate. Recently, wereported the hydrogen bonding assembly of poly(N-vinylpyrrolidone) (PVPON) and alkyne-modified poly(L-glutamic acid)(PGAAlk) multilayers on particles stabilized via click chem-istry by using a bis-azide cross-linker. Upon removal of thesacrificial template with hydrofluoric acid and the disruption ofhydrogen bonding at physiological pH, biodegradable, pH-responsive PGA capsules were obtained.28 These capsules werealso loaded with the anticancer therapeutic doxorubicin (DOX)by incorporating the polymer-drug conjugate PGAAlkþDOX inthe multilayer shell. Control over drug dose and position wasachieved by controlling the number and position of incorporatedPGAAlkþDOX layers. We have also reported the assembly of low-fouling PVPONAlk capsules based on the hydrogen bonding as-sembly of alkyne-modified poly(N-vinyl pyrrolidone) (PVPONAlk)and poly(methacrylic acid) (PMA), followedby click cross linkingand removal of the sacrificial template and hydrogen-bondedPMA.47

Herein, we show that PVPONAlk can be combined withPGAAlk in a modular approach to assemble stratified multilayercapsules of PGAAlk/PVPONAlk with biodegradable reservoirlayers and nondegradable barrier layers exhibiting defineddegradation profiles. This presents an important step toward time-controlled degradation and multidrug release. This approach canbe utilized as a simple yet effective technique for control overdegradation and the release of multilayer films and ultimately therelease of drug layers. Two distinct approaches can be used to

Scheme 1. Assembly of Biodegradable (PVPON/PGAAlk)m (Green, A) and Nondegradable (PVPONAlk/PMA)n (Red, B) Multilayer Filmsa

aBy combining both systems, stratified hybrid capsules with engineered and tailored degradation profiles can be constructed.

(31) Malam, Y.; Loizidou,M.; Seifalian, A.M.Trends Pharmacol. Sci. 2009, 30,592–599.(32) Passarella, R. J.; Spratt, D. E.; van der Ende, A. E.; Phillips, J. G.; Wu,

H. M.; Sathiyakumar, V.; Zhou, L.; Hallahan, D. E.; Harth, E.; Diaz, R. CancerRes. 2010, 70, 4550–4559.(33) Schartl, W. Nanoscale 2010, 2, 829–843.(34) Delcea, M.; Yashchenok, A.; Videnova, K.; Kreft, O.; Moehwald, H.;

Skirtach, A. G. Macromol. Biosci. 2010, 10, 465–474.(35) Kim, B. S.; Smith, R. C.; Poon, Z.; Hammond, P. T. Langmuir 2009, 25,

14086–14092.(36) Chandrawati, R.; Hosta-Rigau, L.; Vanderstraaten, D.; Lokuliyana, S. A.;

Stadler, B.; Albericio, F.; Caruso, F. ACS Nano 2010, 4, 1351–1361.(37) Hosta-Rigau, L.; Stadler, B.; Yan, Y.; Nice, E. C.; Heath, J. K.; Albericio,

F.; Caruso, F. Adv. Funct. Mater. 2010, 20, 59–66.(38) Stadler, B.; Chandrawati, R.; Price, A. D.; Chong, S. F.; Breheney, K.;

Postma, A.; Connal, L. A.; Zelikin, A. N.; Caruso, F.Angew. Chem., Int. Ed. 2009,48, 4359–4362.(39) Kulygin, O.; Price, A. D.; Chong, S. F.; St€adler, B.; Zelikin, A. N.; Caruso,

F. Small 2010, 6, 1558–1564.(40) Malcher, M.; Volodkin, D.; Heurtault, B.; Andre, P.; Schaaf, P.; Mohwald,

H.; Voegel, J. C.; Sokolowski, A.; Ball, V.; Boulmedais, F.; Frisch, B. Langmuir2008, 24, 10209–10215.(41) Volodkin, D.; Arntz, Y.; Schaaf, P.; Moehwald, H.; Voegel, J. C.; Ball, V.

Soft Matter 2008, 4, 122–130.(42) Volodkin, D.; Mohwald, H.; Voegel, J. C.; Ball, V. J. Controlled Release

2007, 117, 111–120.(43) Kreft, O.; Prevot, M.; Moehwald, H.; Sukhorukov, G. B. Angew. Chem.,

Int. Ed. 2007, 46, 5605–5608.(44) Kreft, O.; Skirtach, A. G.; Sukhorukov, G. B.; Moehwald, H. Adv. Mater.

2007, 19, 3142–3145.

(45) Garza, J. M.; Jessel, N.; Ladam, G.; Dupray, V.; Muller, S.; Stoltz, J. F.;Schaaf, P.; Voegel, J. C.; Lavalle, P. Langmuir 2005, 21, 12372–12377.

(46) Garza, J. M.; Schaaf, P.; Muller, S.; Ball, V.; Stoltz, J. F.; Voegel, J. C.;Lavalle, P. Langmuir 2004, 20, 7298–7302.

(47) Kinnane, C. R.; Such, G. K.; Antequera-Garcia, G.; Yan, Y.; Dodds, S. J.;Liz-Marzan, L. M.; Caruso, F. Biomacromolecules 2009, 10, 2839–2846.

DOI: 10.1021/la104232r 1277Langmuir 2011, 27(4), 1275–1280

Ochs et al. Article

achieve this control. First, as shown in Scheme 1, biodegradablePGAAlk capsules (green) and nondegradable low-foulingPVPONAlk films (red) can be constructed using a modularhydrogen-bonding approach, followed by covalent stabiliza-tion using reducible or nonreducible bis-azide cross-linkers. Moreimportantly, stratified hybrid films of (PVPON/PGAAlk)mand (PVPONAlk/PMA)n can be constructed by combiningPGAAlk and PVPONAlk components (A and B in Scheme 1).Furthermore, the degradation of PGAAlk capsules can be tunedby the choice of the bis-azide cross-linker. The facile design ofbiodegradable capsules with defined properties provides a power-ful approach to the assembly of therapeutic carrier systemstailored to specific biomedical applications.

Experimental Section

Materials. Poly(L-glutamic acid, sodium salt) (PGA, Mw=50000 - 70000 Da), poly(N-vinyl pyrrolidone) (PVPON,Mw=55000 Da), and poly(methacrylic acid) (PMA, 30 wt %, Mw=15000 Da) were purchased from Sigma-Aldrich. Nonporoussilica particles (5 wt % suspensions, average diameter 3.25 (0.18 μm) were obtained from Microparticles GmbH (Berlin,Germany). Protease (from Streptomyces griseus, 4.6 units/mg ofsolid) was obtained from Sigma-Aldrich. Gibco phosphate-buff-ered saline (PBS) at pH7.2 was used as received.Unless otherwisespecified, all other chemicals were purchased fromSigma-Aldrichandusedas received. The pHof the solutionswasmeasuredwith aMettler-Toledo MP220 pH meter. High-purity water with aresistivity greater than 18 MΩ cm was obtained from an in-lineMillipore RiOs/Origin water purification system.

Polymer Synthesis. Details of the synthesis of PGA withalkyne functionality (PGAAlk), poly(N-vinyl pyrrolidone-ran-propargyl acrylate) (PVPONAlk), a reducible (N,N0-(dithioethane-2,1-diyl)bis(1-azido tetraethyleneglycol acetamide)) (bis-azide),and a nonreducible cross-linker (bis-azido dodecaethyleneglycol) are described in our previous publications.28,47 The respec-tive structures can be found in Supporting Information Figure S1.

Multilayer Assembly on Silica Particles. In a typicalexperiment, 100 μLof the particle stock solutionwas washed withwater at pH 4. A PVPON solution (400 μL, 1 mg mL-1) wasadded to the pellet, and adsorption was allowed to proceed for20 min with constant shaking of the mixture. Afterwards, theparticles were isolated by centrifugation (1300g for 1 min) andthe supernatant was removed. In a washing step, 400 μL of pH 4water was added. The particles were then redispersed and centri-fuged again, and the washing procedure was repeated twice.For adsorption of the next layer, 400 μL of the PGAAlk solution(1 mg mL-1) was added, followed by the same washing protocol.(PVPON/PGAAlk) multilayer films were prepared by repeatingthis process until the desired number of layers was obtained.Afterwards, the PGAAlk layers were cross-linked overnight byadding a mixture of 300 μL of bis-azide (1 mg mL-1), 100 μL ofsodium ascorbate (3.6 mg mL-1), and 100 μL of copper sulfate(1.44 mg mL-1). Sacrificial polymer PVPON was subsequentlyremoved by raising the pH to disrupt hydrogen bonding (threewashes with PBS pH 7.2).

Stratifiedhybridmultilayer filmswere assembled by combiningthe procedures for the assembly of (PVPON/PGAAlk) and(PVPONAlk/PMA) as described in the Results and Discussionsection.28,47 Hollow capsules were formed by dissolving thetemplate cores using hydrofluoric acid (HF) buffered to pH 5with ammonium fluoride.48 (Caution! Hydrofluoric acid andammonium fluoride are very toxic. Extreme care should betaken when handling HF solution, and only small quantitiesshould be prepared.) At least three washing steps (4500g for 5min, replacing the supernatant with fresh water) were appliedto remove the HF and isolate the capsules for analysis.

Capsules were fluorescently labeled with Alexa Fluor 488(AF488) by incubating them in a solution containing AF488-azide (0.03 μg mL-1), sodium ascorbate (0.72 mg mL-1), andcopper sulfate (0.29mgmL-1) for 24 h. Click-labeled capsuleswere then washed with PBS three times to remove excess dyeand click reagents.

Capsule Degradation. Cross-linked (PGAAlk)m(PVPONAlk)nstratified capsules were washed into PBS and counted on the flowcytometer. Capsules (5 �105) were then incubated with proteasesolution at the indicated concentration in PBS at 37 �C. Similarly,samples stabilized with the reducible bis-azide cross-linker were alsodegraded by a solution of dithiothreitol (DTT, 2 mgmL-1 in PBS).A control sample of capsules was incubated in PBS only. Aliquotswere taken after the indicated periods of time, and the fluorescencewasmonitoredwith flow cytometry. The degradationof thePGAAlk

click capsules was also observed with fluorescence microscopy.

Methods.Flowcytometrymeasurementswere carried out onaPartec CyFlow Space (Partec GmbH, Germany) flow cytometerat an excitation wavelength of 488 nm. Data were analyzedaccording to the procedure outlined previously.49 Differentialinterference contrast (DIC) and fluorescence images were takenon an inverted Olympus IX71 microscope equipped with a DICslider (U-DICT, Olympus) with a 60� objective lens (OlympusUPFL20/0.5NA,W.D. 1.6). ACCD camerawasmounted on theleft-hand port of the microscope. A tungsten lamp was used forDIC images. Fluorescence images were illuminated with an Hgarc lampusing aUF1032 filter cube. For atomic forcemicroscopy(AFM) and transmission electron microscopy (TEM) measure-ments, 1 μL of a concentrated capsule solution was placed on aclean silicon wafer slide (or TEM grid) and allowed to dry. AFMscans were carried out with an MFP-3D Asylum Researchinstrument in ac mode using ultrasharp SiN gold-coated canti-levers (NT-MDT). TEM analysis was carried out with a TecnaiTF30 microscope operated at 200 kV.

Results and Discussion

In this study, different ways of tuning the degradation kineticsof biodegradable and stratified click capsules were investigated.Stratified hybrid capsules of a biodegradable (PGAAlk) module(denoted as A in Scheme 1) and a nondegradable (PVPONAlk)component (denoted as B in Scheme 1) were assembled using themodular hydrogen bonding LbL approach to construct capsuleswith defined degradation profiles. The sacrificial hydrogen-bondingpartners (PVPON and PMA) used in the assembly on particletemplates were removed after capsule formation and washing intoPBS. PGAAlk and PVPONAlk with alkylation degrees of approxi-mately 20% and 1%, respectively, were used throughout this study.It was previously reported that PGAAlk capsules exhibit pH-responsive reversible swelling upon exposure to solutions of pH 2and 11.28 However, we observed that the pH-responsive swel-ling can be tuned via different degrees ofmodificationwith alkynemoieties, leading to a 30% greater responsiveness for PGAAlk

capsules with only 10% alkyne modification (Supporting Informa-tion and Figure S2).

First, capsules were assembled with (PGAAlk)4 as a biodegrad-able base system and different numbers of (PVPONAlk)n=1-4 asthe outermost (barrier) layers. Capsules of these and otherarchitectures produced from ∼3-μm-diameter particle templateswere analyzed by optical microscopy, AFM and TEM, as shownfor representative (PGAAlk)2(PVPONAlk)2(PGAAlk)2(PVPONAlk)2capsules (A2B2A2B2 architecture) in Figure 1A-D. Particletemplates (∼3 μm diameter) were used to facilitate the character-ization andmonitoring degradation of the subsequently produced

(48) Wang, Y. J.; Caruso, F. Chem. Mater. 2006, 18, 4089–4100.(49) Johnston, A. P. R.; Zelikin, A. N.; Lee, L.; Caruso, F.Anal. Chem. 2006, 78,

5913–5919.

1278 DOI: 10.1021/la104232r Langmuir 2011, 27(4), 1275–1280

Article Ochs et al.

capsules. However, smaller template sizes for the preparation ofcapsules with sub-100 nmdiameter can also be used.50-52Negligibleaggregation was observed for capsules in solution (Figure 1A,B),and upon drying for AFM and TEM, the capsules collapsed andfolded as expected (Figure 1C,D). The individual layer thicknessof the hybrid capsules was extracted from AFM profiles and wasfound to be approximately 2 nm per layer, which is within therange observed for similar systems.20,24,28,47

The capsules were then fluorescently labeled using AF488-azide (synthesis details in the Experimental Section) and in-cubated with 1 mgmL-1 protease under physiological conditions(PBS, 37 �C), and their degradation was monitored by flowcytometry. As shown in Figure 2, the degradation of capsuleswith A4Bn=1-4 architecture at the utilized (high) enzyme concen-trations was complete after approximately 2, 4, and 9 h for 1-3barrier layers of PVPONAlk, respectively. The longer degradationtime with increasing PVPONAlk layer number is likely caused byan increase in the barrier thickness to the diffusing enzyme, thuscausing an incubation period before degradation commences.The degradation of stratified capsules was also monitored withfluorescence microscopy for samples taken before and afterthe addition of protease (Supporting Information Figure S3).No capsules, but a strong background fluorescence of thedegraded capsule solution was observed after exposure to protease(Figure S3B). Capsules with 4 barrier layers did not degrade, evenafter extended periods of time (up to 20h). Even after the additionof fresh enzyme solution (4 mg mL-1), no degradation of thesecapsules was observed. This suggests that the cleaving enzymecannot permeate the degradable PGAAlk layers (within the time-frames studied) because of a sufficiently thick barrier layerof PVPONAlk. Furthermore, these data show that the degrada-tion kinetics of stratified (PGAAlk)4(PVPONAlk)1-4 capsules canbe tailored, which is potentially promising in regard to the sus-

tained release of therapeutic cargo coupled to PGA. Previously,we reported the assembly of PGAAlkþDOX capsules with controlover drug dose and position.28,53 Multiple drugs could be loadedas covalently bound polymer-drug conjugates to the PGAAlk

reservoir layers of thehereinpresented systems.The release profile ofthese drugs could potentially be further tuned by the position andnumber of PVPONAlk barrier layers, enabling cascade reactionsand the multistep release of different drugs from a single multi-functional system. Although drug release studies are beyond thescope of this study, as a first step toward this concept we assembledstratified multilayer films of different architectures to study theeffect of the position and the number of nondegradable filmcomponents on capsule degradation.

Capsules of different architectures were also assembled toinvestigate the influence of barrier layer position on the degrada-tion profile. Fluorescently labeled, stratified capsules of A2B2A2B2,B2A2B2A2, and (BA)4 architectures aswell as degradable (A8) andnondegradable (B8) control capsule samples were exposed to a0.5 mg mL-1 protease solution under physiological conditions.A lower concentrationwas used than in the earlier study to closelyobserve the smaller effects of capsule architecture on the degrada-tionprofile. Figure 3 suggests that stratified capsuleswith PGAAlk

as the outermost layer degraded faster than those terminated with

Figure 1. (PGAAlk)2(PVPONAlk)2(PGAAlk)2(PVPONAlk)2 (i.e.,A2B2A2B2) capsules obtained from 3-μm-diameter particle tem-plates, as observed by (A) differential interference contrastmicros-copy, (B) fluorescencemicroscopy (PGApostlabeledwithAF488),(C)AFM(areasof50� 50μm2and5� 5μm2(inset)), and(D)TEM.

Figure 2. Degradation profiles of stratified (PGAAlk)4-(PVPONAlk)n=1-4 (i.e., A4B1-4) capsules of various architecturesassembled from 3-μm-diameter particle templates after incuba-tion with protease under physiological conditions. Samples of allcapsules were incubated in PBS as a control; i.e., the average isplotted for the four capsule types (A4B1-4) in PBSwithout protease.The effect of the number of terminating (PVPONAlk) layers on thedegradation kinetics was monitored by flow cytometry. PGAwaspostlabeled with AF488.

Figure 3. Degradation profiles of stratified (PGAAlk)m-(PVPONAlk)n capsules of various architectures assembled from3-μm-diameter particle templates after incubation with proteaseunder physiological conditions. Samples of all capsules were incu-bated in PBS as a control; i.e., the average is plotted for the fivecapsule types (as denoted in the legend) in PBS without protease.The effect of capsule architecture on the degradation kinetics wasmonitored by flow cytometry. PGAwas postlabeled with AF488.

(50) Gittins, D. I.; Caruso, F. Adv. Mater. 2000, 12, 1947–1949.(51) Gittins, D. I.; Caruso, F. J. Phys. Chem. B 2001, 105, 6846–6852.(52) Schneider, G.; Decher, G. Nano Lett. 2004, 4, 1833–1839.

(53) Yan, Y.; Ochs, C. J.; Such, G. K.; Heath, J. K.; Nice, E. C.; Caruso, F. Adv.Mater. 2010, early view, DOI: adma.201003162.

DOI: 10.1021/la104232r 1279Langmuir 2011, 27(4), 1275–1280

Ochs et al. Article

PVPONAlk. Furthermore, (BA)4 capsules were found to degradeslightly faster than B2A2B2A2 architectures, which is attributed totheir degradation being hindered by only one barrier layer be-tween the degradable PGAAlk layers. Similar degradation kineticswere expected for the A2B2A2B2 architecture (Figure 3) and A4B2

capsules (Figure 2) because in both cases the degradable PGAAlk

layers are protected by the same number of nondegradablePVPONAlk layers. However, the significantly slower degradationof A2B2A2B2 (complete degradation after ∼30 h) was attributednot only to the lower enzyme concentration but also to the existenceof a second barrier layer of PVPONAlk delaying the degradation ofPGAAlk. The importance of the barrier layer position was furtherdemonstrated by comparing sample A2B2A2B2 to A4B4. Al-though containing the same number of degradable and nonde-gradable layers, A2B2A2B2 capsules disassembled after 30 h in a0.5 mg mL-1 protease solution whereas A4B4 capsules (comparewith Figure 2) were not degraded even after an extended period oftime (up to 20 h) and at significantly higher enzyme concentra-tions (up to 4 mg mL-1). This indicates that, even with the samenumber of PGAAlk layers in the multilayer film, degradation alsodepends on the position of the PVPONAlk barrier layers. Allcontrol samples followed the expected kinetics, with A8 beingdegraded after approximately 5 h and B8 as well as the controlsamples in PBS remaining at a stable fluorescence level, indicatingthat no degradation occurred for these samples (over 50 h).

In the second part of this study, we report an additionalapproach to controlling the degradation behavior of PGAAlk

capsules. LbL capsules can be engineered with properties thatallow them to release their cargo at a specific location through thechoice of the polymeric components or the stabilization approachused. For example, capsules stabilized with a disulfide containingbis-azide cross-linker are expected to degrade in a reducingenvironment, such as that found in the intracellular cyto-solic space.54-56 PGA, on the other hand, is susceptible toenzymatic degradation in the lysosome,57,58 making PGA poly-mer-drug conjugates suitable materials for the delivery of(hydrophobic) drugs requiring intracellular release.59,60 Usingthe modular approach presented herein, the degradation of thePGAAlk backbone by lysosomal enzymes can be combined withthe degradability of a reducible bis-azide cross-linker. Thus,synergistic effects (faster degradation) may be achieved in vivo,and different cellular environments (lysosome and cytosolic space)could be targeted with the same capsule system. To investigatewhether PGAAlk capsules could successfully be degraded usingdifferent mechanisms, PGAAlk films were cross-linked using eithera reducible or a nonreducible bis-azide cross-linker (respectivestructures in Figure S1). Instead of the stratified (PGAAlk)m-(PVPONAlk)n capsules introduced above, PGAAlk capsules wereused in this study to simplify the experiments by investigating aone-component system. However, we expect that the concept ofcross-linker variation is also transferable to the stratified capsulesdescribed above. The resulting capsules proved to be degradableby enzymatic cleavage (0.1 mg mL-1 protease solution in PBS,37 �C), as reported previously28 and as shown in Figure 4A.

Although capsules cross-linked with the nondisulfide linker werestable under reducing conditions (2 mg mL-1 dithiothreitol(DTT) in PBS, 37 �C, Figure 4A), those covalently stabilized bya disulfide containing bis-azide were disassembled in a reducingenvironment after approximately 2 h (Figure 4B). This demon-strates that capsules produced using modular hydrogen bondingassembly canbe selectively degradedvia either thePGAbackboneor the cross-linker. The kinetics of DTT-mediated degradationare in agreementwith the results observed for PVPONAlk capsulescross-linked with either the reducible or nonreducible bis-azide.47

For the same number of layers, PVPONAlk capsules stabilizedwith the reducible cross-linker were completely degraded in2 mg mL-1 DTT after 2 h (unpublished data). Consequently,the degradation of PGAAlk capsules after exposure to DTT(Figure 4B) can be attributed to the cleavage of the disulfidecross-linker. Similarly, PVPONAlk capsules were degraded after 4h of exposure to a physiological concentration (5 mM) of anatural thiol-containing peptide (glutathione (GSH)), suggestingthat PGAAlk capsules may also be cleaved by GSH. Other clickcross-linkers exploring enzyme-cleavable, hydrolytically labile, orpH-responsive cleavage sites are the focus of current studies.

Conclusions

We have presented several approaches to tailoring the degra-dation properties of biodegradable polymer capsules based onPGAAlk. It was demonstrated that a combination of degradablePGAAlk modules with a nondegradable PVPONAlk componentcould be used to produce stratified hybrid capsules. The numberand position of nondegradable PVPONAlk layers were found toaffect the degradation kinetics. The degradation could be con-trolled between 2 and 9 h by the addition of 1-3 terminatingPVPONAlk layers, with further deposition resulting in nonde-gradable capsules. Furthermore, capsules terminated with biode-gradable PGAAlk layers were found to degrade faster than thosewith nondegradable PVPONAlk as the outermost layer. Addi-tionally, the degradation of PGAAlk capsules could be controlled

Figure 4. Degradation profile of (PGAAlk)5 capsules assembledfrom 3-μm-diameter particle templates after incubation in DTT,protease, or PBS (control sample) as monitored by flow cytometryfor films cross-linkedwith (A) anonreducible bis-azide cross-linkeror (B) a reducible bis-azide cross-linker.

(54) Becker, A. L.; Zelikin, A.N.; Johnston, A. P.R.; Caruso, F.Langmuir 2009,25, 14079–14085.(55) Zhang, L.; Liu, W. G.; Lin, L.; Chen, D. Y.; Stenze, M. H. Biomacromol-

ecules 2008, 9, 3321–3331.(56) Saito, G.; Swanson, J. A.; Lee, K.D.Adv. DrugDelivery Rev. 2003, 55, 199–

215.(57) Nair, L. S.; Laurencin, C. T. Prog. Polym. Sci. 2007, 32, 762–798.(58) Chiu, H. C.; Kopeckova, P.; Deshmane, S. S.; Kopecek, J. J. Biomed.

Mater. Res. 1997, 34, 381–392.(59) Li, C. Adv. Drug Delivery Rev. 2002, 54, 695–713.(60) Li, C.; Yu, D. F.; Newman, R. A.; Cabral, F.; Stephens, L. C.; Hunter, N.;

Milas, L.; Wallace, S. Cancer Res. 1998, 58, 2404–2409.

1280 DOI: 10.1021/la104232r Langmuir 2011, 27(4), 1275–1280

Article Ochs et al.

by the choice of cross-linker. PGAAlk capsules stabilized witha reducible bis-azide cross-linker could be degraded in 2 h inDTT solution, whereas capsules cross-linked with a nonreduciblebis-azide were stable for up to 8 h. Biodegradable click capsuleswith tunable responsive, low-fouling, and degradation proper-ties are promising candidates for applications in the biomedicalfield.

Acknowledgment.This work was supported by the AustralianResearch Council under the Federation Fellowship (F.C.) and

Discovery Project schemes. Martin van Koeverden is acknowl-edged for help with the capsule swelling studies.

Supporting Information Available: Structures of the re-ducible and nonreducible bis-azide cross-linkers. Tailoringof the pH-responsive swelling behavior of PGAAlk capsulesafter exposure to pH 2 and 11 media. Degradation of strati-fied (PGAAlk)m(PVPONAlk)n capsules as monitored by fluo-rescencemicroscopy. This material is available free of chargevia the Internet at http://pubs.acs.org.