Designing Hyaluronic Acid-Based Layer-by-Layer Capsules as aCarrier for Intracellular Drug Delivery

Anna Szarpak,† Di Cui,† Frederic Dubreuil,† Bruno G. De Geest,‡ Liesbeth J. De Cock,‡

Catherine Picart,§ and Rachel Auzely-Velty*,†

Centre de Recherches sur les Macromolecules Vegetales (CERMAV-CNRS), BP53, 38041 Grenoblecedex 9, France, Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent

University, Harelbekestraat 72, 9000 Ghent, Belgium, Minatec, Grenoble Institute of Technology andLMGP, 3 parvis Louis Neel, F-38016 Grenoble Cedex, France

Received November 14, 2009; Revised Manuscript Received January 11, 2010

Polyelectrolyte microcapsules were prepared by the layer-by-layer assembly of hyaluronic acid (HA) and apolycationic polymer, poly(allylamine) (PAH) or poly(lysine) (PLL). The influence of the polycationic partneron the morphology, stability, permeability properties, and enzymatic degradation of microcapsules was thoroughlyanalyzed. It was found that these properties could be tuned by shell cross-linking. Confocal microscopy studiesof cellular uptake of the capsules showed that the polyelectrolyte shells remain stable outside the cells but readilybreak open once internalized by cells, suggesting their potential as carrier for intracellular drug delivery.

Introduction

The use of polyelectrolyte microcapsules is currently beingstudied as a method to deliver drugs to cells in a controlledand selective way. Such systems, having a shell thickness inthe nanometer range, are made by the layer-by-layer (LbL)assembly1 of oppositely charged polyelectrolytes on a sacrificialtemplate, followed by its decomposition. Several advantagesof polyelectrolyte capsules have been noted. First, they can beprepared under mild conditions which allow encapsulation oflarge amounts of fragile biologically active molecules.2-4

Second, by varying the layer number and the multilayercomposition, a high degree of functionality within their shellcan be obtained.5-9 Third, the capsule surface can be designedwith a wide variety of functionalities, such as lipids,10 poly-(ethylene glycol),11 antibodies,12 or sugars.13 Such surfacemodifications allowed to obtain biofunctional capsules havingthe potential not only to resist to nonspecific adsorption ofproteins11 but also to target cells specifically.12 Moreover,cellular uptake of capsules was demonstrated.12,14-20 In thiscontext, several efforts have been made toward the developmentof smart capsules that are able to release their content insidethe cell in response to external or internal stimuli. It was thusshown that after internalization by cells, capsules made ofnonbiodegradable synthetic polymers (poly(diallyl dimethylam-monium chloride), PDADMAC, and poly(styrene sulfonate),PSS) remained intact but, upon light illumination, could beopened and thereby release the cargo from their cavity.17,19 Thislight-activated release was due to local disruption of the shellresulting from the heat produced by irradiated gold nanoparticlesthat were incorporated during multilayer assembly construction.Another strategy consisted in the synthesis of degradablepolyelectrolyte capsules containing polyelectrolytes that can bedegraded either enzymatically (polypeptides or polysaccharides)

or through hydrolysis.14,21-23 After cellular uptake, suchcapsules were degraded over approximately 24 h, contrary tocapsules made of the synthetic polyelectrolytes which remainedquasi-intact.14 Regarding enzymatic degradation, it was alsoshown that the wall properties of capsules can be readilychanged upon varying enzyme concentrations.23 In view of drugdelivery applications, polyelectrolyte capsules templated onporous inorganic cores such as calcium carbonate (CaCO3) andmesoporous silica have recently emerged as promising carriersfor macromolecular drugs such as protein antigens and DNA,providing high encapsulation efficiency.24-30

In a previous paper, we reported on the synthesis of capsulesfrom hyaluronic acid (HA), a highly hydrated natural polysac-charide which plays an important structural and biological rolein the living organisms.31 In spite of the property of HA to formsoft hydrogel-type multilayered films and its weak polyacidcharacter, we developed conditions for the construction of stablehollow capsules in combination with poly(allylamine hydro-chloride) (PAH), a synthetic polycation.31 Such capsules didnot show cytotoxicity after contact at a high concentration within vitro cultured myoblast cells. However, they contain PAHas a nonbiodegradable component which impairs their futureuse in a clinical setting. In this work, we focused on the designof fully biodegradable capsules based on HA with the purposeto create carriers for intracellular drug delivery. Poly(L-lysine)(PLL), a biocompatible and biodegradable polypeptide, was usedas polycation in combination with HA as polyanion to formmultilayer microcapsules. We first investigated the morphologyand stability properties of these microcapsules under physi-ological conditions using several complementary techniques.Because disruption of the multilayer was observed for HA/PLLcapsules, we investigated a possible route to improve the shellstability by chemical cross-linking. These chemical modifica-tions and their effect on the permeability properties of thecapsule membrane in the absence and in the presence ofendogenous enzymes are described in the second section of thispaper. Finally, we also report on the intracellular uptake andintracellular fate of the capsules by macrophages. Our data pave

* To whom correspondence should be addressed. Phone:+33(0)476037671.Fax:+33(0)476547203.E-mail:rachel.auzely@cermav.cnrs.fr.

† CERMAV-CNRS, affiliated with Universite Joseph Fourier, andmember of the Institut de Chimie Moleculaire de Grenoble.

‡ Ghent University.§ Minatec, Grenoble Institute of Technology and LMGP.

Biomacromolecules XXXX, xxx, 000 A

10.1021/bm9012937 XXXX American Chemical Society

the way for the development of tailor-made capsules based onhyaluronic acid.

Experimental Section

Materials. Hyaluronic acid under the sodium salt form, having amolar mass Mw of 820 × 103 g/mol, was a gift from ARD (Pomacle,France). We selected this HA sample for the preparation of capsulesaccording to our previous work31 showing that diffusion of the initialpolyelectrolyte layers in the porous carbonate core became limited whenthe HA molar mass was equal to or higher than 820 × 103 g/mol.Poly(allylamine hydrochloride) (Mw ∼ 70 × 103 g/mol), poly(L-lysinehydrobromide) (Mw ∼ (15-30) × 103 g/mol), dextran samples labeledwith fluorescein isothiocyanate with molar mass of 4 × 103, 500 ×103, and 2000 × 103 g/mol (dextranFITC-4, -500 and -2000), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES sodium salt), tr-is(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), ethylene-

diaminetetraacetic acid (EDTA), calcium chloride (CaCl2), sodiumcarbonate (Na2CO3), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-ride (EDC) were purchased from Sigma-Aldrich-Fluka. LysoTrackerRed was purchased from Invitrogen. All chemicals were used withoutany further purification. The water used in all experiments was purifiedby a Millipore Milli-Q Plus purification system, with a resistivity of18.2 MΩ cm.

Capsule Preparation. Microcapsules were prepared using calciumcarbonate microparticles as a sacrificial template. CaCO3 microparticleswere synthesized from solutions of CaCl2 and Na2CO3 as reported inthe literature.4,32 The CaCO3 microparticles were coated using the layer-by-layer technique, by incubating them at a concentration 2% (w/v)2

in an aqueous solution (0.15 M NaCl, pH 6.5) of HA (Cp ) 5 g/L) andPAH (Cp ) 2 g/L) or PLL (Cp ) 2 g/L). After shaking for 10 min, themicroparticles were collected by centrifugation and the residualnonadsorbed polyelectrolyte was removed by washing twice with anaqueous solution of 0.01 M NaCl (pH 6.5).

After deposition of 4.5 layers, the CaCO3 core was removed bytreatment with a 0.1 M EDTA solution (pH 7.5). To avoid mechanicaldamages of “soft” polyelectrolyte shells, the dissolved ions resultingfrom the decomposition of CaCO3 were removed by dialysis againstpure water, using spectra Por dialysis bags with a molar mass cut offof 6-8 kDa.

Cross-Linking of the HA/PAH Multilayers. Chemical cross-linkingof the multilayer shells was performed by activation of the carboxylicacid groups of HA using the water-soluble carbodiimide, EDC, andsulfo-NHS in 0.15 M NaCl.33 Various EDC concentrations (50, 100,200 mM) were used, whereas the concentration of sulfo-NHS was keptconstant at 50 mM. The core-templated HA/PAH mulilayers wereincubated in the EDC/sulfo-NHS solution (pH 6.5) overnight. The coreswere dissolved by shaking the particle dispersion in 0.1 M EDTA (pH) 7.5).

Isothermal Titration Calorimetry (ITC). ITC experiments werecarried out on a Microcal VP-ITC titration microcalorimeter (Northamp-ton, U.S.A.). All titrations were made in 0.01 M Tris-HCl buffer pH7.4 with 0.15 M NaCl at 25 °C. The reaction cell (V ) 1.45 mL)contained the PAH solution (Cp ) 2 g/L corresponding to [PAH] )21.3 mM, calculated from the average molecular weight of the repeatingunit) or PLL solution (5 g/L corresponding to [PLL] ) 25 mM). Aseries of 10 injections of 10 µL from the computer-controlled 300 µLmicrosyringe at an interval of 10 min of the solution of HA (Cp ) 3g/L corresponding to [HA] ) 7.4 mM) were performed into thepolycation solution while stirring at 300 rpm at 25 °C. Under suchconditions, endothermic heat was produced after each injection of HAand the magnitude of the released heat was nearly constant for bothpolyanion/polycation systems, as the chains of HA established amaximum number of interactions with the PLL and PAH chains. Indeed,the polycations are in large excess compared to HA, even after the teninjections. The amount of heat produced per injection was calculatedby integration of the area under individual peaks by the instrumentsoftware, after taking into account heat of dilution. The ∆H value forPAH/HA and PLL/HA complexation given in this paper is an averagevalue of the ∆H values derived from the ten peaks.

Figure 1. Scanning electron microscopy images showing the surfaceof spherical CaCO3 microparticles (A) before and (B,C) after deposi-tion of 4.5 HA/PLL and 4.5 HA/PAH bilayers. The first and outermostdeposited layer is HA.

Figure 2. Film growth monitored in situ by QCM-D for (PLL/HA; 0)and (PAH/HA; 9) films. (a) Difference in the frequency shifts ∆fmeasured at 15 MHz after each polycation and polyanion deposit.(b) Film thickness as a function of the number of deposited layersdeduced from the QCM-D data for the two films.

Figure 3. Scanning electron microscopy images showing (HA/PLL)4.5

(A) and (HA/PAH)4.5 (B) dried hollow capsules. The first and outermostdeposited layer is HA. Holes can be distinguished on the HA/PLLshell as indicated by an arrow.

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Films Characterization by Quartz Crystal Microbalance withDissipation Monitoring (QCM-D). The (PAH/HA)i and (PAH/PLL)i

film buildup (where i denotes the number of layer pairs) was followedby in situ quartz crystal microbalance (QCM with dissipation monitor-ing, D300, Qsense, Sweden).34 The quartz crystal was excited at itsfundamental frequency (about 5 MHz, ν ) 1) as well as at the third,fifth and seventh overtones (ν ) 3, 5, and 7 corresponding to 15, 25,and 35 MHz, respectively). Changes in the resonance frequencies ∆fand in the relaxation of the vibration once the excitation is stoppedwere measured at the four frequencies. As the silica coated quartz crystalis negatively charged, depositions always started with the positivelycharged polyelectrolyte. The QCM-D data have been analyzed using aVoigt based model35 implemented in the Qtools software (Q-Sense)using predetermined values for the film density (1009 kg/m3), bufferdensity (1000 kg/m3), and viscosity (1 mPa.s). Three parameters (filmthickness, viscosity, and shear modulus) are then deduced.

Confocal Laser Scanning Microscopy (CLSM). Capsules suspen-sions were observed with a Leica TCS SP2 AOBS (Acoustico OpticalBean Splitter) confocal laser scanning system and, an invertedfluorescence microscope equipped with an oil immersion objective lens63×. FITC-labeled dextran samples were visualized by excitation ofthe fluorochrome with a 488 nm argon/krypton laser and the emittedfluorescence was collected between 497 and 576 nm, precisely definedby the AOBS.

Scanning Electron Microscopy (SEM). Drops of capsules suspen-sions were deposited onto copper stubs and allowed to air drying. Thesamples were sputtered with Au/Pd and observed in secondary electronimaging mode with a Jeol JSM6100 microscope using an acceleratingvoltage of 8 kV. For high resolution SEM analysis, the specimens werecoated by 2 nm of electron beam evaporation carbon and observed insecondary electron imaging mode with a Zeiss ultra 55 FEG-SEM(CMTC-INPG, Grenoble) at an accelerating voltage of 3 kV, using anin-lens detector.

Incubation with FITC-Labeled Dextrans. Suspensions of HA/PAHand HA/PLL capsules in 0.02 M MES buffer (pH 6.5) were leftovernight at room temperature. The FITC-labeled dextran samples weredissolved in the same buffer at a concentration of 2 mg/mL. Forpermeability tests in the presence of salt, NaCl was added into MESbuffer to obtain a concentration of 0.15 M. Typically, 20 µL of a

dextranFITC solution was mixed with 20 µL of capsule suspension on aglass slide. After 20 min, the capsules were observed by CLSM.

The images were analyzed using Leica Confocal Software by themeasurement of the light emitted by the capsule interior (Iint) andsurrounding solution (Iext). The permeability coefficient (Iint/Iext × 100%)was estimated as an average value from 7-10 capsules.

Cell Culture Experiments. RAW 264.7 mouse macrophages werepurchased from ATCC and cultured in DMEM (Invitrogen) supple-mented with 10% FBS, 1% penicillin-streptomycin (Invitrogen) and1% glutamine. The cells were seeded onto round sterile cell culturechambers, equipped with a microscopy coverslip as bottom, and allowedto attach overnight. Subsequently, the cells were incubated with capsulesfor 2 h followed by lysosomal staining with LysoTracker Red accordingto the manufacturer’s instructions.

Results and Discussion

1. Synthesis of HA/PAH and HA/PLL HollowCapsules. HA/PAH and HA/PLL multilayer capsules werefabricated by using CaCO3 microparticles as a template and theoptimal conditions reported previously.31 Thus, high Mw HA(Mw ) 820 × 103 g/mol) and the polycation (PAH or PLL)were alternatively deposited at a concentration of 5 and 2 g/Lin water containing 0.15 M NaCl at pH 6.5, respectively.Deposition of the polyanions and polycations on calciumcarbonate cores could be demonstrated by SEM analysis. Ascan be seen in Figure 1, the surface of CaCO3 particles coatedwith 4.5 bilayers of HA/PLL or HA/PAH becomes smootherthan the initial porous particles. It exhibits in both cases bumpyislets which, in the case of the surface of HA/PAH coatedparticles, appear to be larger and coalesce to form an almostuniform film. Such differences in film pattern suggest differentrate of multilayer growth depending on the polycation. To getmore information about the multilayer build-up, the step-by-step deposition of HA/PAH and HA/PLL under identicalpolymer concentrations as for capsule synthesis was followedon a solid planar substrate by QCM-D (Figure 2). As previouslyreported,31 HA/PAH film growth is exponential in our working

Figure 4. SEM (Top) and AFM (bottom) images of (HA/PAH)4.5 (A,C) and (HA/PLL)4.5 (B,D) dried capsules cross-linked by means of 200 mMEDC.

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conditions (0.15 M NaCl at pH 6.5). Actually, HA/PAH filmshave already been described as exponentially growing films ina previous study performed at the same ionic strength for a HAsample of intermediate molecular weight (4 × 105 g/mol),36

but a linear growth was reported at low ionic strength conditions(0.01 M NaCl).37 Exponential growth could be related here tothe diffusion of PAH within the film architecture, as previouslyobserved for films containing HA in combination with polysac-charides such as chitosan (CHI)38 or polypeptides, such asPLL.39 In comparison with HA/PAH, HA/PLL film growth wasslower, presumably due to the fact that we are still in the earlystages of the buildup. Indeed, for these films, several investiga-tors have shown a transition in growth rates between a precursorregime and “the regular” growth regime.40-43 The number ofbilayers for which the crossover occurs (∼8 layer pairs for HA/PLL34) was found to coincide with the stage at which the filmsurface is fully covered with polyelectrolyte complex.41 Incontrast, it can be assumed from Figure 2 that in the case ofHA/PAH, full surface coverage is achieved after adsorption of3 or 4 bilayers.

We also measured by ITC the complexation enthalpy for bothpolyelectrolyte couples to investigate if differences in filmgrowth could be attributed to different affinities between thepolyelectrolytes. The complexation enthalpy (∆H) values de-rived from ITC experiments (cf. Supporting Information, FigureS1) performed with our polyelectrolyte pairs were respectivelyof 560 ( 55 J/mol for PAH/HA and 1479 ( 145 J/mol forPLL/HA complexes in 0.01 M Tris-HCl buffer with 0.15 MNaCl (pH 7.4). Of note, the entropy gain is not measurable byITC but it is accepted that polyelectrolyte complexation isassociated with a entropy gain (positive ∆S) due to the releaseof associated counterions and water.44 For the polyelectrolytecomplexation to take place, the free energy of interaction ∆G(∆G ) ∆H - T∆S) has to be negative, which indicates that, inour case, the entropic contribution has to drive the formationof the complexes.

Interestingly, Laugel et al.45 recently reported a correlationbetween the nature of the growth process and the heat ofcomplexation between the polyanions and the polycationsconstituting the multilayer film. These authors found that anendothermic process is rather characteristic of an exponentialfilm growth, whereas a strongly exothermic process correspondsto a linear growth regime. Thus, based on the enthalpic valuesobtained, both HA/PAH and HA/PLL films are expected togrowth exponentially, which was indeed observed by Laugelet al.45 However, from the comparison between the ∆H values(higher for HA/PLL than for HA/PAH, i.e., less favorable), onecan assume that the film buildup for the HA/PLL system maybe slower. Indeed, this is what we observed experimentally byQCM-D (Figure 2).

As carbonate particles showed a high tendency to aggregatewhen coated with more than five HA/PAH or HA/PLL bilayers,the production of capsules by core dissolution was performedfrom carbonate cores coated with 4.5 bilayers. In a first step,we imaged by SEM the morphologies of the different capsulesobtained after core dissolution by treatment with a 0.1 Mbuffered EDTA solution (Figure 3). As previously reported inour first work on HA/PAH capsules,31 the capsules are collapsedafter drying with a diameter of ∼5 µm, providing evidence ofcore removal. On the other hand, the HA/PLL capsules appearmuch smaller (∼2.5 µm). These observations suggest that theHA/PLL exhibit an important shrinkage, which is maybe relatedto HA/PLL complexation. Capsules shrinkage was previouslyreported by Mauser et al.,46 when Ca2+ was added to capsules

made of poly(methacrylic acid) (PMA) and PAH. Such aphenomenon was attributed to the competitive binding of Ca2+

to PMA -COO- groups, thereby reducing the electrostaticinteractions between PMA and PAH, which stabilize themultilayer wall. This may also partly explain the reducedstability of the HA/PLL capsules after treatment with EDTA.Indeed, due to the fact that HA/PLL electrostatic interactionsare weaker than HA/PAH, we can assume that Ca2+ ions mayact as a competitor of PLL by forming a HA/Ca2+ complex,causing precipitation of HA and capsule shrinkage.

Based on these results and the hypothesis, we decided in thenext step to cross-link the HA/PLL shell to increase the stabilityof capsules after core dissolution.

2. Manipulating the Shell Properties by ChemicalCross-Linking. The capsule shells were cross-linked followingthe procedure already developed for HA-based polyelectrolytemultilayers on planar surfaces33 and HA/PAH capsules31

assembly, consisting in an amine-carboxyl coupling reaction.The carboxylate groups of HA are thus converted by reactionwith 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-chloride in combination with N-hydroxysulfosuccinimide intoactivated esters, which can then be reacted with the aminegroups of the polycation to form amide bonds. The cross-linkingreactions were performed on HA/polycation multilayers depos-ited on CaCO3 particles under mild conditions (pH 6.5), allowingto maintain the polyelectrolyte complex integrity. The multilayercoated particles were incubated in freshly prepared EDC/sulfo-NHS solutions overnight at room temperature followed by coredissolution and washing with water. Considering that theconcentration of the coupling agent can influence the degree ofcross-linking and, hence, the film’s mechanical properties,47,48

different concentrations of EDC (50, 200, and 400 mM) weretested for HA/PAH and HA/PLL capsules. Structural andmorphological changes after cross-linking were analyzed bymeans of FTIR-ATR spectroscopy. Several modifications of theFTIR-ATR spectrum of HA/PLL capsules after cross-linkingcould be observed (cf. Supporting Information, Figures S2 andS3). The carboxylic peaks of HA (at 1400 and 1610 cm-1)decreased, while the amide I and amide II bands (at ∼1670and ∼1540 cm-1, respectively) increased.

The cross-linked capsules were further subjected to SEM andAFM characterizations to reveal their morphological changes.Figure 4A,B shows that the cross-linked HA/PAH and HA/PLLcapsules have smooth surface and a compact thin wall. Theyexhibit the typical folds and creases observed for syntheticpolyelectrolyte multilayer capsules49 as well as biopolyelectro-lytes capsules50 (Figure 4C,D). The swelling of capsule wallthus seems to be restricted. Of note, all the cross-linked capsulesexhibited well formed spherical shapes with a size of ∼5 µmin aqueous solution (see Figure 5B,D). Thus, the shrinkage ofHA/PLL capsules could be prevented by cross-linking themultilayer assembly. One can also notice that cross-linkedcapsules have lower tendency to agglomerate (Figure 5B,D) thanthe un-cross-linked (Figure 5A,C).

By using AFM, the thickness of the dried polyelectrolyteshells could be estimated according to the method reported byLeporatti et al.51 They are reported in Table 1. It can be noticedthat the thickness values are in the same order of magnitude ofthose measured by ellipsometry on planar films in the drystate.37,52 Of note, higher thickness values were obtained in thewet state by QCM-D on planar films, which is consistent withthe fact the films are hydrated due to the presence of HA.

The permeability of the un-cross-linked and cross-linkedcapsules toward hydrophilic dextrans was next investigated

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(Figure 5). These experiments revealed that shell cross-linkinghas a drastic effect on the capsule’s permeability. In fact, thediffusion of a low molar mass dextranFITC was very high insidethe un-cross-linked capsules, while the cross-linked ones(prepared using an EDC concentration of 200 mM) were almosttotally impermeable.

Furthermore, we studied the effect of the cross-linkerconcentration on the shell permeability for dextran moleculesof different molar masses (4 × 103, 500 × 103, 2000 × 103

g/mol; Figure 6). Interestingly, the permeability of HA/PAHand HA/PLL cross-linked capsules toward dextranFITC-500 anddextranFITC-2000 remains very low (equal or near to zero) forall cross-linked capsules incubated in MES buffer without NaCl.In fact, only the smallest molecule investigated, DextranFITC-4,partially diffused into the capsules interior, with permeabilityvalues depending on the amount of EDC used. The capsuleswere only permeable for the lowest concentration of EDC used(50 mM) while they were impermeable for the highest ones(200 and 400 mM). Of note, at a low concentration of couplingagent (50 mM), the diffusion of dextranFITC-4 molecules washigher in the case of HA/PLL (32%) than HA/PAH capsules(∼25%).

Also and interestingly, the cross-linked capsules containingclosed pores in 0.02 M MES buffer were found to open pores

by increasing salt concentration (0.15 M NaCl) in the surround-ing medium. This dramatically increased diffusion of moleculesinto capsules, especially that of dextranFITC-4. As previouslyreported,31 treatment of a HA/PAH film on a planar surface byEDC at a concentration of 200 mM leads to a cross-linkingdegree of 0.35. This indicates that many charged groups maybe still involved in electrostatic interactions within the multilayerwall. This salt effect can be thus related to a weakening of theelectrostaticbindingbetweentheoppositelychargedpolyelectrolytes.53,54

Thus, based on these data, it seems possible to tune thepermeability of the capsules by shell cross-linking. Cross-linkingendows the capsules with a stronger ability to reduce the extentof capsule wall swelling. Our results are in line with others fromthe literature. A reduction of permeability in the shells aftercross-linking was observed for the (PAH/PSS)5 capsules whichin the native form were permeable for dextran at Mw 460 ×103 g/mol and became impermeable after treatment withglutaraldehyde as a cross-linking agent.49 Cross-linking ofalginate in the (alginate/chitosan)5 capsule shells with calciumions decreased the insulin release rate.55 It was also shown thatthe drug release could be tailored by the cross-linking densityof dextran-based multilayer capsules prepared by click chem-istry.56

3. Enzyme-Responsive Shell Permeability. Owing to thepotential applications of HA capsules as drug delivery systems,

Figure 5. Effect of shell cross-linking on the morphology andpermeability toward dextranFITC-4 of (HA/PAH)4.5 and (HA/PLL)4.5

capsules. Top: CLSM images of un-cross-linked (A,C) and cross-linked (B,D) (HA/PAH)4.5 and (HA/PLL)4.5 capsules incubated indextranFITC-4 solution (2 g/L in 0.02 M MES buffer, pH 6.5). The twoleft images corresponding to the HA/PAH capsules and the two rightimages, to the HA/PLL capsules. Bottom: comparison of the perme-ability properties of (HA/PAH)4.5 and (HA/PLL)4.5 capsules in 0.02 MMES buffer (pH 6.5). Permeability is expressed as the ratio offluorescence intensities of the capsules interior (Iint) and surroundingsolution (Iext) 20 min after mixing capsules and solutions of dextranFITC-4. Cross-linking was performed using EDC at a concentration of 200mM. The average value was taken from 10 capsules.

Table 1. Comparison of the Thickness in the Dry State ofCross-Linked Capsules and Planar Films Prepared from HA/PAHand HA/PLL

thickness

multilayer

capsulesa inthe dry state(4.5 bilayers)

planar films inthe dry state(5 bilayers)b

planar films inthe wet state(4 bilayers)c

HA/PAH (50 ( 13) nm ∼40 nm37 ∼140 nmHA/PLL (26 ( 5) nm ∼20 nm52 ∼100 nm

a Thicknesses were measured by AFM. b Thicknesses were measuredby ellipsometry. c Thicknesses were measured by QCM-D.

Figure 6. Ratio of the fluorescence intensities of the capsules interior(Iint) and surrounding solution (Iext) 20 min after mixing capsules andsolutions containing dextranFITC with different molar masses (4 × 103g/mol (O), 500 × 103g/mol (0), and 2000 × 103g/mol (4) for HA/PLL(A) and HA/PAH (B) capsules at different cross-linker concentration.Experiments were performed either without NaCl (open symbols) orwith 0.15 M NaCl (filled symbols) in a buffer (0.02 M MES buffer, pH6.5).

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we investigated their degradation by a tissue enzyme. Cross-linked and un-cross-linked capsules were thus incubated at 37°C in the presence of testicular hyaluronidase (Hase) atconcentrations in the range of 10-500 U/mL in MES buffer

(Figure 7). To exclude a possible effect of the temperature jumpfrom 25 to 37 °C, we verified that it did not affect that wallpermeability (Figure 7D). The contact with Hase at concentra-tions of 10 and 50 U/mL did not lead to noticeable changes ofHA capsules (data not shown). In the presence of 500 U/mLHase, only the un-cross-linked HA/PLL capsules with initialshrunken morphology lose their structural stability; the shellwere disintegrated (Figure 7B). Increase in permeability of HA/PLL capsules can be related to more “porous” structure of thesecapsules allowing the enzyme to diffuse deeper into polymercomplex/shell, resulting in a higher degradation of the multi-layer. This higher sensitivity of the HA/PLL capsules may bedue to weaker interactions between the polyelectrolytes com-pared to the HA/PAH systems. Moreover, it can be noted thatthe concentration used for HA/PLL degradation is in the samerange of magnitude of concentrations required for the degrada-tion of planar multilayer films33,48 and gels.57,58 Degradationof cross-linked capsules evidenced in Figure 7D shows thatpermeability of HA/PAH shells does not change after contactwith enzyme contrary to the HA/PLL capsules of whichpermeability increased ∼10 folds. However, the cross-linkedcapsules did not change their spherical shape after contact withenzyme (Figure 7C). These results may suggest that the shellsare only partially degraded due to the presence of covalent bondsbetween polyelectrolyte partners which hinder accessibility toglycosidic bonds. In a recent work, Lee et al.22 found that BSAis released faster from cross-linked HA/PLL as soon as Haseconcentration is equal to or greater than 10 U/mL. However,these capsules, having a diameter of ∼16 µm, were preparedunder different conditions from a HA sample of Mw 64 × 103

g/mol and were cross-linked with EDC only, a reaction whichis much less efficient than in the presence of sulfo-NHS.33 Itcan be noted that the Hase concentration in the human serumis of the order of 2.6 U/mL.58 However, the physiological

Figure 7. Effect of hyaluronidase on the permeability toward dex-tranFITC-4 of (HA/PAH)4.5 and (HA/PLL)4.5 un-cross-linked and cross-linked capsules (200 mM EDC). Top: transmission images of un-cross-linked (A,B) and cross-linked (C) (HA/PLL)4.5 microcapsules inthe absence (A) and in the presence of hyaluronidase (B, C) at aconcentration of 500 U/mL. In both cases, capsules were left overnightat 37 °C in 0.02 M MES buffer. Bottom: ratio of the fluorescenceintensities of the capsules interior (Iint) and surrounding solution (Iext)20 min after mixing cross-linked capsules and MES buffer containingdextranFITC-4.

Figure 8. Confocal microscopy images of (A) cross-linked HA/PAH and (B) cross-linked HA/PLL capsules after 2 h coincubation with RAWmouse macrophages. Capsules are stained green fluorescent using HAFITC, while the cellular lysosomes are stained using LysoTracker Red.The left pane gives the overlay of the green and red channel, the middle pane is the DIC channel and the right pane is the overlay of green, red,and DIC. Colocalization between the green and red channel is observed as a yellow/orange color.

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concentrations of Hase depend on the location in the body andother factors such as thermal degradation and attack by freeradicals are also responsible for the degradation of hyaluronicacid. The greater resistance of our HA-based capsules to theenzymatic hydrolysis could be due to the formation of a compactnetwork with a lower permeability to enzymes as suggested forchemically cross-linked HA hydrogels.57-59 The accessibilityof HA in the multilayer assembly may thus depend on the molarmass of HA chains and on the cross-linking extent. Thisassumption regarding the influence of the molar mass issupported by degradation studies performed on un-cross-linkedHA/CHI planar films,48 which showed that assemblies made ofCHI with Mw ) 100 × 103 g/mol are more resistant todegradation by Hase (500 U/mL) than those containing CHIwith Mw ) 5 × 103 g/mol.

This work thus demonstrated that the stability and perme-ability properties of the HA capsules can be tuned by controlledchemical modifications of the shell.

4. In Vitro Capsule-Cell Interactions. To assess whetherthe cross-linked HA-based capsules can be taken up byphagocyting cells, RAW mouse macrophages were cultured for2 h in the presence of capsules and observed by confocalmicroscopy imaging. For visualization purpose and to assessthe microcapsules intracellular fate, the capsules were stainedusing HAFITC (green fluorescence) while the cellular lysosomeswere stained with LysoTracker Red (a fluorescent dye emittingred fluorescence, which selectively accumulates in acidic cellularvesicles). As depicted in Figure 8, both HA/PAH and HA/PLLcapsules are intact while being outside cells and become largelydeformed and break-up when internalized within the cells.

Moreover, for all phagocyted capsules, complete colocaliza-tion between green capsule fluorescence and red lysosomalfluorescence is observed, indicating that the capsules end-up inlysosomal vesicles. This is in accordance with previous reportsby the Parak16 and De Geest14,60 groups. However, the deforma-tion kinetics of the internalized capsules (few hours) is remark-ably faster compared to earlier reported capsules. Bedard et al.21

studied the deformation upon cellular uptake of syntheticnondegradable PSS/PDADMAC capsules. It was shown that,after 15 h, capsules deformation occurred, however the capsulesdid not break. De Geest14 and De Koker15,25 investigatedintracellular degradation in vitro and in vivo of capsules basedon dextran sulfate and poly(L-arginine), but in contrast to theHA-based capsules in this paper it took more than 24 h beforecapsule deformation and degradation occurred. The fast capsulebreak-up observed in our present study clearly offers perspectiveto use the capsules as a intracellular delivery carrier that stablyencapsulates its payload in the extracellular space but whichreadily opens upon cellular uptake.

Conclusions

In this work, we designed fully biodegradable capsules basedon hyaluronic acid and poly(L-lysine). By investigating themorphology and permeability properties of capsules, we assessedwhich parameters are important to obtain capsules that are stableunder physiological conditions.

Contrary to capsules containing the synthetic polycation PAH,capsules containing PLL exhibited strong shrinkage after coredissolution. By chemically cross-linking the shell before tem-plate dissolution, we could prevent this shrinkage and stablecapsules with limited permeability to relatively small hydrophilicmolecules (dextranFITC-4) were obtained. Regardless of thechemical cross-linking, the HA/PLL capsules were still respon-

sive to a tissue enzyme, hyaluronidase, showing higher perme-ability toward dextranFITC-4. Such a behavior suggests potentialto control drug release from these capsules through a biodeg-radation process. Finally we demonstrated that both HA/PLLas well as HA/PAH capsules become rapidly (i.e., within 2 h)internalized in endo/lyso-somatic vesicles upon incubation within vitro cultured macrophages. Subsequently, we observed aremarkably fast intracellular rupturing of the capsules, whileextracellular capsules remained intact. Such fast intracellularopening of capsules without external triggering has, to the bestof our knowledge, not yet been reported and could offer adistinct advantage for the delivery of, for example, proteinantigen, where the encapsulated payload becomes readilyavailable for processing in an early stage of endo/lyso-somaticacidification.

Acknowledgment. We gratefully acknowledge FredericCharlot at CMTC-INPG, Grenoble, for his help with SEMobservations using the Zeiss ultra 55 FEG-SEM. This work hasbeen supported by “Agence Nationale pour la Recherche” ANR-07-NANO-002 to R.A. and C.P. C.P. is a Junior Member ofthe “Institut Universitaire de France” whose support is gratefullyacknowledged. D.C. and A.S. gratefully acknowledge theMENRT and the E.C. (contract number MEST-CT-2004-503322of Sixth Framework Programme), respectively, for their thesisgrant in CERMAV. B.G.D.G. acknowledges the FWO Vlaan-deren for a postdoctoral scholarship. L.J.D.C. acknowledges theIWT for a Ph.D. scholarship.

Supporting Information Available. Thermograms obtainedfrom the calorimetric titration of PLL and PAH with HA, FTIR-ATR spectra of (HA/PLL)4.5 and (HA/PAH)4.5 hollow driedcapsules without cross-linking and cross-linked with 50, 200,and 400 mM EDC. This material is available free of charge viathe Internet at http://pubs.acs.org.

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