Spontaneous Loading of Positively Charged Macromolecules

Spontaneous Loading of Positively Charged Macromolecules into Alginate-Templated Polyelectrolyte MultilayerMicrocapsulesHuiguang Zhu,*,†Rohit Srivastava,†and Michael J.McShane†,‡Institute for Micromanufacturing and Biomedical Engineering Program,Louisiana Tech University,911Hergot Avenue,Ruston,Louisiana71272Received March4,2005;Revised Manuscript Received May3,2005A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings.A calcium ion chelator, EDTA,was used to free the Ca2+-cross-linked alginate hydrogel within{poly(allylamine hydrochloride)/ poly(styrene sulfonate)}4({PAH/PSS}4)coating,allowing partial release of alginate.The retention of alginate in{PAH/PSS}4microcapsule was confirmed by FTIR spectroscopy and confocal microscopy.Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino,and peroxidase)into alginate-templated microcapsules,which showed the loading occurred in<2min for dextran-amino and<10min for peroxidase,respectively.A high loading efficiency of25µg peroxidase in∼1.0×107microcapsules(2.5pg POx/capsule)was achieved with a low concentration of peroxidase loading solution(10µg/mL).This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.1.IntroductionThe development of layer-by-layer(LbL)self-assembly on colloidal templates has inspired considerable attention in biotechnology and promises solutions to many problems in the fields of drug delivery,biosensors,and microreactors, as well as bioseparations.1This potential is especially strong for the application of polymeric microcapsules,2where polyelectrolyte multilayer walls can not only concentrate, shield,and protect bioactive molecules in a defined volume, but also create a specially defined microenvironment.A number of recent papers have reported on explorations of macromolecule encapsulation in polyelectrolyte capsules.3-7 Typically,materials can be introduced into these microcap-sules through two procedures:encapsulating material within the template core,followed by polyelectrolyte multilayer deposition on the spheres to create the walls;or loading materials into a preformed polyelectrolyte shell.However, a persistent challenge is the loading of desired materials such as enzymes into capsules with high efficiency and stable retention,while preserving their biological activity. Numerous recent attempts have been made to load macromolecules into capsules,using alternative methods to stabilize the encapsulated materials,such as in situ“ship-in-a-bottle”polymerization of various monomers inside the capsules,3changing the pH of the bulk solution to alter the permeability of the multilayer wall,4changing the solvent to keep the molecules from leaching out of the capsules,5 and cross-linking the multilayer wall to make it less permeable for species already loaded into capsules.6There has also been a report of“spontaneous”enzyme deposition into melamine formaldehyde(MF)-templated microcapsules.7 It is believed that the MF oligomers formed charged complexes with polyelectrolyte layer when MF cores were decomposing,such that the charged complexes attracted enzymes into the capsules.Though these previous findings have proved that the property of capsule permeability can be changed by solvent or pH exchange,most of these processes are reversible and depend on the exterior environ-ment,properties that may be undesirable in certain situations. It is also necessary to consider the biocompatibility of the core materials when used for biomedical applications.For some applications,such as biosensors,a permanent adjust-ment of the physicochemical properties of the wall archi-tecture is desired,so that encapsulated molecules are stably retained in a biocompatible matrix.Alginate hydrogels,ionically cross-linked in the presence of multivalent cations,have been extensively investigated due to their biocompatibility.The earliest work could be traced back to as early as1960s.8Alginate encapsulation has been reported as suitable for many biological components including cells,9owing to the relatively inert aqueous environment within the matrix,the mild room-temperature encapsulation process,and high gel porosity allowing high diffusion rates of macromolecules.10Since alginate contains carboxylic acid groups on polyguluronate units,alginate*Corresponding author:Phone:(318)257-5127.Fax:(318)257-5104. Email:hzhu@.†Institute for Micromanufacturing.‡Biomedical Engineering Program.2221Biomacromolecules2005,6,2221-222810.1021/bm0501656CCC:$30.25©2005American Chemical SocietyPublished on Web06/02/2005microspheres exhibit negative surface charge,11allowingthem to be used as negatively charged templates forpolyelectrolyte LbL assembly.The polyelectrolyte multilayerwall can therefore provide a protective shell for encapsulatedbiomolecules,prolonging the life-span of alginate micro-spheres in a biological environment.Multilayer coatingsinvolving chitosan/alginate,poly(lysine)/alginate,and otherpolyelectrolyte coatings have been proven for use as barriermembranes for alginate microspheres to slow release ofencapsulated macromolecules.12The coatings also slow thedecomposition of alginate microspheres relative to uncoatedalginate microspheres in PBS or biological environments.13This makes the structures more mechanically stable,whichmay be important for long-term applications.Due to theseattractive characteristics,alginate gel beads have beeninvestigated as vehicles for enzyme encapsulation.In addition to enabling deposition of nanofilm surfacecoatings,the negatively charged alginate may be used toattract oppositely charged molecules.On the basis of previousfindings with charged MF cores,7it was hypothesized thatthe excess negative charge on alginate molecules inside thepolyelectrolyte coating could serve as a driving force toattract positively charged molecules for high-efficiencyencapsulation;an important feature of alginate is the superiorbiocompatibility to MF particles for biomedical applications.To explore the application of alginate microspheres ascharged matrixes,which is called“electrostatic sponge”,astudy of this promising spontaneous loading process forpositively charged molecules such as enzymes into biocom-patible alginate hydrogel-templated polyelectrolyte multilayercapsules was performed.The ability of these systems toachieve high efficiency encapsulation from low-concentrationloading solution was investigated,and the dependence onmolecular weight and charge was tested.2.Experimental Section2.1.Materials.Sodium alginate(low viscosity,250cps,Mw12k-80k),calcium chloride,sorbitan trioleate(SPAN85),and polyoxyethylene sorbitan trioleate(TWEEN85)were all purchased from Sigma-Aldrich.OmniSolv2,2,4-Trimethylpentane(isooctane)was purchased from EMDChemicals Inc.Sodium poly(styrene sulfonate)(PSS,MW ∼70kDa),poly(allylamine hydrochloride)(PAH,Mw∼70kDa)were obtained from Sigma-Aldrich.N-(3-dimethy-laminopropyl)-N′-ethylcarbodiimide hydrochloride(EDC),N-hydroxysulfosuccinimide sodium salt(NHSS),and1,6-diaminohexane were purchased from Fluka.Ethylenedi-aminetetraacetic acid disodium salt dihydrate(EDTA),FITC-dextran(77kDa,FD),peroxidase(POx),fluorescein isothio-cyanate(FITC,isomer I),and rhodamine B isothiocyanate(RITC)were obtained from Sigma-Aldrich.Dextran-amino(500kDa)was purchased from Molecular Probes.Forconfocal fluorescence microscopy,POx was labeled withRITC in pH8.5sodium bicarbonate buffer and purifiedthrough a SephadexTM G-25M column(Amersham Phar-macia Biotech AB).PAH and dextran-amino were labeledwith RITC in pH8.5sodium bicarbonate buffer andprecipitated in acetone according to the amine labeling protocol from molecular probes.All chemicals were reagent grade and used as received.2.2.Instrumentation and Measurement.An ultrasoni-cator(Cole Palmer,CPX-750,750W total power)was used to disperse water droplets in the oil for the production of alginate microspheres.The size distribution and number of alginate microspheres were measured with a Coulter counter (Beckman Coulter Z Series)using a100µm aperture.Fourier transform infrared spectroscopy(Nexus470FTIR)was used to analyze the unmodified alginate microspheres and the {PAH/PSS}-coated alginate microcapsules.The assembly of polyelectrolyte layers on alginate microspheres was moni-tored by electrophoretic mobility measurements(ZetaPlus Zeta Potential Analyzer,Brookhaven Instrument Corp.). UV-vis spectra were obtained using a Lambda45UV/Vis spectrometer(PerkinElmer).Confocal micrographs were taken with a Leica TCS SP2equipped with a63×oil-immersion objective.2.3.Synthesis of Alginate-FITC Derivative.Sodium alginate(120mg)was mixed with EDC/NHSS(50mg/30 mg)for the activation of carbonyl groups on alginate in pH 5.0sodium acetic buffer for30min,followed by addition of1,6-diaminohexane(60mg)for another4h.The mixture was precipitated in2-propanol to remove unreacted diamine. The alginate-amine derivative was reacted with FITC(0.5 mg)in pH8.5sodium bicarbonate solution for4h and precipitated in acetone.The resulting alginate-FITC deriva-tive,used to facilitate imaging with fluorescence microscopy, was mixed with sodium alginate solution and made into microspheres using the following process.2.4.Fabrication of Alginate Hydrogel Microspheres. Alginate hydrogel microspheres were prepared by an emul-sification technique modified from Wan et al.14In short,50 g3wt%sodium alginate aqueous solution was dispersed in75g isooctane containing1.7g of SPAN85using the ultrasonicator at60%power for5min.A solution containing 0.9g of TWEEN85in5g of isooctane was then added to the emulsion and ultrasonicated at the same power for another 5min to achieve stable water/oil emulsion droplets.After this,20mL of aqueous solution containing10wt%of calcium chloride was added to allow formation of ionic cross-links while stirring for20min.The microspheres were then rinsed three times with DI water by successive centrifugation cycles and stored in DI water at room temperature.2.5.LbL Assembly of PAH/PSS on Alginate Micro-spheres.The sodium poly(styrene sulfonate)(PSS)and poly-(allylamine hydrochloride)(PAH)solutions used for alter-nating adsorption of{PAH/PSS}multilayers on alginate microspheres were prepared at a concentration of2mg/mL in DI water containing0.1M CaCl2.For deposition of each coating,1mL of polyelectrolyte solution was added to a microcentrifuge tube containing100µL of microsphere suspension(∼106spheres).Adsorption was allowed to proceed for20min,after which the suspension was centrifuged to separate the spheres from remaining un-adsorbed polyelectrolyte.The microspheres were then triple-rinsed with DI water by successive centrifugation cycles.The process was repeated for the oppositely charged polyelec-2222Biomacromolecules,Vol.6,No.4,2005Zhu et al.trolyte,and then alternated until a total of four bilayers of {PAH/PSS }films were obtained.2.6.De-Cross-Linking of Ca 2+-Alginate Hydrogel in Alginate-Templated Microcapsules.Alginate microspheres coated with four bilayers of {PAH/PSS }were suspended in 1mL DI water in a microcentrifuge tube.EDTA solution (100µL of 0.1M)was added to de-cross-link the Ca 2+-alginate ionic hydrogel by chelating the calcium ions.Following 30min incubation at room temperature with mild shaking,the microcapsules were centrifuged and rinsed with DI water three times.The supernatants were dried on a ZnSe crystal and analyzed with FTIR spectroscopy to assess the presence of alginate.2.7.Spontaneous Loading of Dextran-Amino and POx into Alginate-Templated Microcapsules.To compare struc-ture-dependent loading properties,alginate spheres with different properties were exposed to solutions containing positively charged macromolecules.Alginate microspheres,alginate microspheres with four bilayers {PAH/PSS }coat-ings,and EDTA-treated alginate-templated {PAH/PSS }4microcapsules were suspended in DI water for loading dextran-amino and pH3.0buffer for loading POx,respec-tively.Dextran-amino and POx were then added into the appropriate suspension,with a final concentration of 10µg/mL and a total volume of 2mL.3.Results and Discussion3.1.Fabrication of Alginate Microspheres and {PAH/PSS }LbL Assembly on Microspheres.Alginate hydrogel microspheres were prepared by an emulsification technique,as illustrated in Scheme 1.Coulter counter measurements revealed that the process resulted in a typical alginate microsphere mean diameter of4.25(0.49µm (N >5000spheres counted).The microspheres were found to be stable in DI water after removal of the surfactant,and exhibited the expected negative surface charge,which therefore could be used for polyelectrolyte LbL assembly to provide protec-tive shells for use in a biological environment.The LbL assembly of polyelectrolyte layers was monitored by electrophoretic mobility measurements.Four bilayers of {PAH/PSS }were deposited on alginate cores,and the surface potential of the microspheres was observed to change regularly from -30mV for alginate and PSS to +40mV for PAH (Figure 1),indicating the formation of the desired {PAH/PSS }4wall architecture.This result demonstrates that the alginate microspheres still have strong negative surface charge after calcium gelation,which makes polyelectrolyte nanofilm deposition onto alginate microspheres straightfor-ward.The polyelectrolyte multilayer wall may serve multiple purposes,including providing a protective shell for encap-sulated biomolecules and stablizing alginate microspheres against dissolution in a biological environment.Confocal microscopy was used to image the FITC-labeled alginate microspheres (Figure 2a)and the spheres with {RITC -PAH/PSS }4coating (Figure 2b),which clearly shows the alginate microspheres fabricated by emulsification technique and the applied polyelectrolyte coating.It was found the size of alginate microspheres were relative uniform fabricated from emulsion technique,and the average diameter of the alginate microspheres is about 4µm in Figure 2.Additionally,the FITC labeled alginate was uniformly distributed before and after {PAH/PSS }4coating.FTIR analysis was performed to determine the composition of alginate microspheres,PAH/PSS mixtures,and alginate/{PAH/PSS }4(Figure 3).It can be seen that the most significant peak in the IR spectrum for alginate (Spectrum a)is at about 1600cm -1,contributed to carbonyl groups on alginate molecules.Spectrum b is the absorbance of PSS/PAH complexes.The bonds at 1115and 1170cm -1are attributed to the symmetric stretching vibration and asym-metric vibration of -SO 2-units,respectively,15while the signals at 1590cm -1and 1470cm -1are attributed to N -H bending and C -H bending,respectively.The peaks at 3400cm -1and 3150cm -1also indicate the presence of -NH 2groups.For alginate/{PAH/PSS }4(Spectrum c),two typical shoulders are present at 1740cm -1(amide I)and 1540cm -1(amide II),suggesting chemical complexation of carboxylic acid groups on alginate with amine groups on PAH.Additionally,the same features from -SO 2-,indicating the presence of PSS,are observed at 1100-1200cm -1.As a result,Spectrum c suggests that alginate/{PAH/PSS }4pro-Scheme 1.Emulsification Technique for the Production of Alginate Microspheres aaInset shows confocal image of FITC-labeled alginate microspheres.Figure 1. -potential measurements for deposition of {PAH/PSS }4on alginate microspheres (n )5).The first measurement (layer 0)is the surface potential of alginate microspheres,after removal of surfactant.Spontaneous Loading of Macromolecules in CapsulesBiomacromolecules,Vol.6,No.4,20052223duces a combining spectra of alginate microspheres and PAH/PSS mixtures.3.2.De-Cross-Linking of Alginate Microspheres with {PAH/PSS }4.Coating EDTA was used as a Ca 2+chelator to free the Ca 2+-cross-linked alginate hydrogel within the {PAH/PSS }4coating,allowing partial release of alginate molecules.It was hypothesized that some alginate would remain complexed with the cationic inner layer of PAH and would not be completely removed from the capsules.FTIR was used to analyze the supernatant after EDTA treatment of the alginate/{PAH/PSS }4capsules (Figure 4).Following EDTA treatment,strong alginate absorbance was observed in the supernatant from the 1st rinsing with DI water.The alginate absorbance decreased significantly after the 2nd rinsing,and was nearly undetectable in the IR spectra after 3rd rinsing.The alginate-templated microcapsules were again observed by confocal microscopy after EDTA treatment and there was no detectable FITC fluorescence (Figure 5a)when using the same laser power setting as in Figure 2.However,FITC-labeled alginate was observed inside of the {PAH/PSS }4microcapsules (Figure 5b)when the laser power was increased to 64times of the initial power.It was found that the alginate could not be completely removed from the {PAH/PSS }4microcapsules and was still uniformly distrib-uted in the capsules,probably because of the positive PAH inner layer.This result verified our hypothesis that the Ca 2+-alginate hydrogel could be decomposed by EDTA treatment within the polyelectrolyte multilayer microcapsules,which therefore allows partial release of alginate molecules from the {PAH/PSS }4microcapsules.Furthermore,the fluores-cence images prove the presence of alginate in the capsules following Ca 2+release;the residual charged matrix was then further investigated for use in electrostatic absorption of cationic molecules from the surrounding solution.3.3.Spontaneous Loading of Dextran-Amino and Per-oxidase into Alginate-Templated Microcapsules.After partial removal of the alginate core,the loading of positively charged molecules into capsules was studied with confocal microscopy.For these experiments,unlabeled alginate was used.Dextran-amino (500kDa)was tagged with RITC (TD500k)to allow tracking of the loading process.FordirectFigure 2.Confocal images of (a)alginate microspheres and (b)alginate microspheres with 4bilayers of {RITC -PAH/PSS }(alginate labeled with FITC (green),PAH labeled with RITC(red)).Figure 3.FTIR spectra of (a)alginate microspheres,(b)PAH/PSS mixture,and (c)alginate microspheres with four bilayers of {PAH/PSS }.Figure 4.FTIR spectrum of the supernatant from the alginate microspheres with {PAH/PSS }4and treated with EDTA after 1-3times of rinsing.Inset shows magnified view of spectra after 2nd and 3rd rinses.2224Biomacromolecules,Vol.6,No.4,2005Zhu et al.comparison,anionic FITC-dextran (Mw 77kDa,FD77k)was used.Figure 6contains images of the capsules exposed to TD500k and FD77k using a concentration of 10µg/mL dextran in each loading solution.Figure 6a,c contains images of the {RITC -PAH/PSS }4capsules prior to addition of the loading solutions.After adding TD500k,the red RITCFigure 5.Confocal images of alginate microspheres with {PAH/PSS }4following EDTA treatment under (a)initial and (b)64×of initial laser power (alginate labeled with FITC (green),PAH labeled with RITC (red)).Figure 6.Confocal images from a loading experiment to compare uptake of positively-and negatively charged macromolecules.Alginate-templated capsules (a)before and (b)after loading positively charged TD500k;(c)before and (d)after exposure to negatively charged FD77k.Spontaneous Loading of Macromolecules in Capsules Biomacromolecules,Vol.6,No.4,20052225fluorescence inside the capsules increased rapidly and reached a high intensity (Figure 6b)within 5min.This result suggests that the positively charged dextran-amino was loaded into alginate-templated capsules with high efficiency very quickly.Furthermore,it can be seen that TD500k was uniformly distributed inside the microcapsules,which verified the residual alginate after EDTA treatment was also uni-formly distributed inside the microcapsules.On the other hand,green fluorescence from the negatively charged FD77k did not reach a measurable level within the capsules even after 24h exposure (Figure 6d).Thus,despite the lower molecular weight (∼7×smaller),it appears that the anionic FD77k cannot penetrate the capsules.Therefore,it is believed that the excess negative charge on alginate in the capsules is the driving force for loading of positively charged macromolecules.A schematic diagram describing this spon-taneous loading procedure of positively charged molecules into alginate-templated microcapsules is shown in Scheme 2.Real-time confocal microscopy was used to further study the loading kinetics for alginate-templated capsules,as well as alginate spheres with and without polyelectrolyte coatings.For these experiments,the final concentration of TD500k added to spheres/capsules/suspensions was 10µg/ing constant laser intensity and PMT gain,images were collected every 15s,and the fluorescence intensity from regions of interest (RoIs)inside optically sectioned capsules was plotted versus time (Figure 7).From these loading results,it can be observed that TD500k was efficiently loaded into all three samples,but with different uptake rates depending upon the material properties.It is clear that TD500k loads into {PAH/PSS }4-coated intact spheres much more slowly (about 43min to reach PMT saturation)than the other two samples (about 2and 13min for alginate-templated capsules and uncoated spheres,respectively).This is expected because of the diffusion barrier presented by both cross-linked alginate and the wall of multilayer film.The de-cross-linking and partial dissolution of alginate not only provides more space but also weakens the structural barrier presented by cross-linked alginate that decreases permeability of capsules.Selected images for capsules at t )0,60,and 120s duringthe loading process are also shown in Figure 8.It can be seen that the dextran molecules were first adsorbed into or onto the polyelectrolyte multilayer wall,then continued to penetrate into the capsule slowly and reach a steady-state level.By reducing the PMT gain,the line-scan of fluores-cence intensity for single capsules proved that TRITC-dextran was uniformly distributed throughout the capsules,as is shown for POx in Figure 9.The charge-driven loading of positively charged molecules into alginate-templated capsules was further verified using POx (P I )8.0)at pH 3.0.POx was also conjugated with RITC for confocal investigation,and the concentration of POx added in solution was 10µg/mL.From Figure 9,it can be seen that POx also rapidly loaded into alginate capsules with high efficiency,reaching a steady-state level in about 10min.Like dextran,the POx molecules were also first adsorbed into or onto the polyelectrolyte multilayer wall and then penetrate into the capsule slowly and reach a steady-state level.Since the primary function of enzymes is catalysis,it is desirable to efficiently load enzyme into microspheres while minimizing the loss of enzyme activity.In this context,Scheme 2.Schematic Diagram of Spontaneous Loading of Positively Charged Macromolecules in Alginate Hydrogel-Templated MicrocapsulesFigure 7.Internal capsule fluorescence intensity for loading of TD500k into (a){PAH/PSS }4-coated alginate microspheres,(b)alginate-templated {PAH/PSS }4microcapsules after EDTA treatment,and (c)uncoated alginate spheres.2226Biomacromolecules,Vol.6,No.4,2005Zhu et al.“efficient”refers to encapsulation of a high percentage of initial enzyme (minimal waste).The loading RITC -POx solution and supernatant after loading were further analyzed with UV -vis spectroscopy to quantify the loading efficiency of POx in alginate microcapsules.Based on the number of microspheres from Coulter counting,and the loaded POx amount calculated from absorbance measurements,it was found that low-concentration loading solution (10µg/mL)may be used to obtain high uptake efficiency:25µg peroxidase in ∼1.0×107microcapsules,which is 2.5pg POx/capsule.This result compares favorably with 35µg peroxidase in 7×106microcapsules (5.0pg POx/capsule),when a loading concentration of 1-3mg/mL was used for MF-templated capsules.7c Therefore,it can be stated that a similar loading efficiency was obtained when using much lower concentration of POx.ConclusionCalcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique,and then used as negatively charged templates to form polyelectrolyte mul-tilayer coatings.Alginate was partially released from {PAH/PSS }4coatings after EDTA treatment,as verified by FTIR spectroscopy and confocal microscopy.Positively charged macromolecules s dextran-amino and peroxidase s were thensuccessfully loaded into EDTA-treated alginate/{PAH/PSS }4polyelectrolyte multilayer microspheres within 10min,as confirmed by real-time confocal microscopy and UV -vis spectroscopy.It was found that the loading efficiency for POx could be as high as 2.5×10-6µg POx/capsule,even when low loading concentration (10µg/mL)was used.The excess negative charge on alginate molecules in microcap-sules was believed as the driving force for material encap-sulation.This spontaneous loading technique results in high loading density for macromolecules,a desired goal for biomedical and biotechnological applications.The alginate matrix may provide a sufficiently gentle and porous scaf-folding to preserve accessibility of the enzyme to substrate as well as biocompatibility.Additional studies are needed to determine the activity and stability of enzymes spontane-ously loaded into alginate capsules,critical features of catalytic devices such as biosensors.Acknowledgment.This work was supported by the National Institutes of Health (#R01EB00739-01)and the National Science Foundation (NIRT 0210298).References and Notes(1)(a)Sukhorukov,G.B.;Donath,E.;Lichtenfeld,H.;Knippel,E.;Knippel,M.;Mo ¨hwald,H.;J.Colloid Surf.A 1998,137,253.(b)Moya,S.;Dahne,L.;Voigt,A.;Leporatti,S.;Donath,E.;Mo ¨hwald,H.J.Colloids Surf.A 2001,27,183.(c)Caruso,F.;Trau,D.;Mo ¨hwald,H.;Renneberg,ngmuir 2000,16,1485.(d)McShane,M.J.;Lvov,yer-by-Layer Electrostatic Self-Assembly.In Encyclopedia of Nanosci.Nanotechnology ;Marcel Dekker:New York,2004;Vol.5.(e)McShane,M.J.;Brown,J.Q.;Guice,K.B.;Lvov,Y.M.J.Nanosci.Nanotech.2002,2,411.(f)Balabushevich,N.G.;Tiourina,O.P.;Volodkin,D.V.;Larionova,N.I.;Sukhorukov,G.B.Biomacromolecules 2003,4(5),1191.(2)(a)Sukhorukov,G.B.;Donath E.;Davis 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A.;Mamedov, A.;Mo ¨hwald,H.;Sukhorukov,G.B.;Nano Lett.2001,1(3),125.(6)(a)Richert,L.;Boulmedais,F.;Lavalle,P.;Mutterer,J.;Ferreux,E.;Decher,G.;Schaaf,P.;Voegel,J.-C.;and Picart,C.Biomacro-molecules 2004,5,284-294.(b)Zhu,H.;McShane,ngmuir 2005,21(1),424Figure 8.Representative confocal images and line-scan data for loading of TD500k into {PAH/PSS }4-coated alginate capsules during the loading process at t )0,60,and 120s.Insets show line-scan of fluorescence intensity for a singlecapsule.Figure 9.Loading of peroxidase into alginate capsules.Inset images showed capsules after peroxidase loading.Spontaneous Loading of Macromolecules in Capsules Biomacromolecules,Vol.6,No.4,20052227。