Colloids and Surfaces A:Physicochem.Eng.Aspects 370 (2010) 28–34Contents lists available at ScienceDirectColloids and Surfaces A:Physicochemical andEngineeringAspectsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r faPreparation of monodispersed polyelectrolyte microcapsules with high encapsulation efficiency by an electrospray techniqueYu Fukui,Tatsuo Maruyama ∗,Yuko Iwamatsu,Akihiro Fujii,Tsutomu Tanaka,Yoshikage Ohmukai,Hideto Matsuyama ∗∗Department of Chemical Science and Engineering,Kobe University,1-1Rokkodai,Nada-ku,Kobe 657-8501,Japana r t i c l e i n f o Article history:Received 29May 2010Received in revised form 11August 2010Accepted 14August 2010Available online 21 August 2010Keywords:Electrospray Microcapsule PolyelectrolyteHigh encapsulation efficiencya b s t r a c tThe preparation of polyelectrolyte microcapsules by electrospray was investigated.When a polyanionic or polycationic aqueous solution was electrosprayed into an aqueous solution containing a polyelectrolyte with the opposite charge,a spherical interface consisting of a polyelectrolyte complex was formed by electrostatic interaction to produce a microcapsule.Alginate/chitosan microcapsules (∼100m)were successfully produced with a narrow diameter distribution (coefficient of variation 4.4%).The diameters of microcapsules were controlled in the range of 80–230m by varying the operating conditions,such as feed rate,working voltage,the distance from needle-to-collector,needle diameter and polyelectrolyte concentrations.We also succeeded in the encapsulation of protein,dextran and a polymeric microsphere within the polyelectrolyte microcapsules with high encapsulation efficiencies (more than 99%).The study of yeast encapsulation reveals that the electrospray technique can encapsulate a physiologically active substrate in the polyelectrolyte microcapsule and maintain its activity.© 2010 Elsevier B.V. All rights reserved.1.IntroductionMicroencapsulation has been widely used in the agricultural,food,cosmetics,pharmaceutical and medical industries [1–3].Along with the emerging demand for new functional microcap-sules,the development of a new microencapsulation technique has been a significant target in the research field to achieve effi-cient encapsulation,a biocompatible microcapsule,a functional microcapsule or a controlled-release strategy [4–6].Emulsifica-tion,spray-drying and coacervation techniques are mostly used for encapsulating food ingredients,proteins,drugs,flavors and liv-ing cells.In the last decade,studies in microfluidics technology have provided a novel strategy for the preparation of microcapsules with narrow size-distributions [7–9].The electrospray technique,which is conventionally used as an ionization technique in mass spectroscopy,has a great potential for microcapsule preparation because of the simplicity of the apparatus,its high productivity and its easy setup.Although the electrospray technique can con-tinuously produce tiny droplets or nanofibers with ease,there has been uncertainty around the production of microcapsules.Bugarski et al.first reported microcapsule preparation using the electro-∗Corresponding author.Tel.:+81788036070;fax:+81788036070.∗∗Corresponding author.E-mail addresses:tmarutcm@crystal.kobe-u.ac.jp (T.Maruyama),matuyama@kobe-u.ac.jp (H.Matsuyama).spray technique and demonstrated the effective preparation of size-controlled microcapsules [10].They and other research groups subsequently reported encapsulation of viable living cells using calcium alginate and the electrospray technique [11–14].These reports employed only calcium alginate as a capsule material,prob-ably due to its safety with living cells.In 1980s,relatively large capsules (∼mm)composed of poly-electrolytes were studied to encapsulate cells and enzymes [15,16].These capsules of millimeter size were prepared simply by adding a charged-polyelectrolyte solution dropwise to another oppositely charged-polyelectrolyte solution.In the last decade,the layer-by-layer method using polyelectrolytes have attracted wide attention as a thin film material because they form a stable and insolu-ble complex with an oppositely charged polyelectrolyte [17].In particular,this method can employ synthetic and natural poly-electrolytes and provide a novel class of functional ultrathin films.The functional properties of polyelectrolyte films (e.g.,controlled release of encapsulated compounds,self-rupturing,biocompatibil-ity and stimuli-responsiveness,etc.)are generally derived from the design and combination of polyelectrolytes [18–21].In some cases,the solubility of the complex formed depends on the pH and the ionic strength of the solution,which was also used for the controlled release of encapsulated compounds or for the stimuli-responsiveness of the thin film.The layer-by-layer method has been also extended to prepare microcapsules using a sacrifice template [22,23].However,the use of a sacrifice template makes it difficult to effectively encapsulate core substrates within a microcapsule.0927-7757/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfa.2010.08.039Y.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects370 (2010) 28–3429The strong electrostatic interaction and fast complex-formation between positively and negatively charged polyelectrolytes require the manipulation of each aqueous solution with another immisci-ble phase(e.g.,a water-immiscible organic solvent or a gas phase) when preparing microcapsules composed of polyelectrolytes.The electrospray technique ejects a droplet to a gas phase at high speed and the droplet falls onto a collector vessel.We expected that the electrospray technique would enable the preparation of monodispersed microcapsules based on cationic and anionic polyelectrolytes with high encapsulation efficiency. The present study reports the preparation of microcapsules using several kinds of polyelectrolytes by the electrospray technique. Most of the experiments were performed using chitosan and alginate as polyelectrolytes because this polyelectrolyte combi-nation was widely applied for capsule production,as can be seen in the following references[24–26].We investigated key parameters of the electrospray technique to control the size of microcapsules and found that the electrospray technique could produce monodispersed microcapsules(around100m in diam-eter)and also encapsulate various materials(biomacromolecules, microparticles and living cells)within the microcapsules with high encapsulation efficiency.2.Experimental2.1.MaterialsSodium alginate,chitosan,acetic acid and sodium hydrox-ide were purchased from Wako Pure Chemical Industries(Osaka, Japan).Poly(sodium4-styrenesulfonate)(PSS,Mw=∼70,000),poly (allylamine hydrochloride)(PAH,Mw=∼56,000),albumin–fluore-scein isothiocyanate conjugate(albumin–FITC),and tetramethyl-rhodamine isothiocyanate–dextran(TRITC–dextran)with average molecular weights of4400,65,000–76,000and155,000were pur-chased from Sigma(St.Louis,MO).Poly(diallyldimethylammonium chloride)solution(PDDA,Mw=40,000,28wt%in H2O)was pur-chased from Polysciences Inc.(Warrington,PA).Greenfluorescent polystyrene microspheres were purchased as a suspension from Duke Scientific(Palo Alto,CA).The microspheres had a diame-ter of1.9m.Yeast extract and peptone were purchased from Becton,Dickinson and Company(Sparks,MD).d-(+)-Glucose was purchased from Nacalaitesque,Inc(Kyoto,Japan).2.2.ElectrosprayThe electrospray(NF-102,MECC Co.,Ogori,Japan)experimen-tal equipment consisted of a syringe pump,a stainless steel needle and a high voltage generator(Fig.1).An anionic or cationic poly-electrolyte aqueous solution was sprayed from a stainless steel needle(cathode)into an aqueous solution containing a polyelec-trolyte with an opposite charge(anode)in a foil-wrapped dish to form polyelectrolyte complex microcapsules.The polyelectrolyte aqueous solution in a dish was stirred continuously and gently (∼100rpm)by a magnetic stirrer bar during electrospraying.Typically,a sodium alginate(1.5wt%)aqueous solution(pH7.5) was sprayed into a chitosan aqueous(0.5wt%)solution(pH3.6) containing200mM acetic acid.The feed rate of the sodium alginate solution was set at0.20mL/h and the working voltage was22.5kV. The distance from the needle to the collector was5.0cm and the inner/outer diameters of a stainless steel needle were130/310m. Microcapsules were prepared under different conditions to control the diameters of microcapsules.After electrospraying,microcap-sules were separated from the chitosan solution by centrifugation at100×g for2min.An invertedfluorescence microscope(Olym-pus,IX71)was employed to observe microcapsules.Based on the microscope images,the diameters of more than100microcapsules Fig.1.Illustration of the experimental setup and photograph of a Taylor cone jet (inset).The Taylor cone was observed when a sodium alginate(1.5wt%)aque-ous solution was sprayed at0.20mL/h into a chitosan(0.5wt%)aqueous solution containing200mM acetic acid at a working voltage of22.5kV and a needle-to-collector distance of5cm.The inner/outer diameters of the stainless steel needle were130/310m.were measured by an image analysis software,WinROOF(Mitani Corp.,Fukui,Japan).PSS/PAH(PSS sprayed into PAH),PAH/PSS,chitosan/PSS and PDDA/PSS were also used for the preparation of microcapsules.The concentrations of PSS,PAH and PDDA were10wt%.The concentra-tion of chitosan solutions was9wt%.Only the chitosan solution contained50mM acetic acid.The pH of PSS,PAH,PDDA and chi-tosan solutions were4.8,1.6,2.6and6.5,respectively.The feed rate was0.20mL/h in each case,the voltage was24.0kV and the needle-to-collector distance was5.0cm.The inner/outer diameters of a stainless steel needle were330/630m.A Taylor cone,formed on the tip of a needle,was observed by a microscope(SKM-2000-PC,Saito Kougaku,Yokohama,Japan).2.3.Encapsulation of polymeric microspheres,protein anddextran and the dextran retention propertiesTo evaluate the encapsulation efficiency,fluorescent micro-spheres,albumin–FITC and TRITC–dextran were used as a core substrate.A sodium alginate(1.5wt%)aqueous solution contain-ing10L/mLfluorescent microspheres,10g/mL albumin–FITC or10g/mL TRITC–dextran was sprayed into0.5wt%chitosan aqueous solution containing200mM acetic acid under typical con-ditions.After spraying,a microcapsule suspension wasfiltered through a nylon-netfilter with a41m mesh(Millipore,NY). For the albumin–FITC encapsulation,thefiltrate was adjusted to pH6.0using sodium hydroxide.Non-encapsulatedfluorescence substrates in thefiltrate were quantified using afluorescence spectrometer(LS-50B,PerkinElmer).Fluorescence microspheres, albumin–FITC and TRITC–dextran were excited at468,495and 555nm,respectively.Fluorescence emissions were detected at 508,521and580nm,respectively.Thefluorescent microsphere-,albumin–FITC-and TRITC–dextran-encapsulated microcapsules were observed using a confocal laser scanning microscopy(CLSM) (FV1000-D,Olympus Co.,Tokyo,Japan).30Y.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects370 (2010) 28–34To determine the dextran retention properties of the microcap-sules,TRITC–dextran-encapsulated microcapsules were collected by centrifugation at100×g for2min and dispersed in3M acetate buffer(pH5.2).Samples were periodically taken from the micro-capsule suspension and centrifuged at100×g.Thefluorescently labeled substrate in the supernatant solution was quantified using afluorescence spectrometer.2.4.Yeast-encapsulated microcapsulesThe yeast Saccharomyces cerevisiae Kyokai No.7was grown in a YPD medium(10g/L yeast extract,20g/L glucose,and20g/L peptone)at30◦C overnight.A sodium alginate(1.5wt%)aqueous solution containing yeast(OD∼0.35)was electrosprayed into a chi-tosan(0.5wt%)aqueous solution containing200mM acetic acid solution under typical conditions.The microcapsules were col-lected by centrifugation at100×g and then immersed in a fresh YPD medium,followed by microscope observation of the yeast growth in microcapsules at25◦C.3.Results and discussionWhen a polyelectrolyte solution is forced through a needle with an electric voltage,the surface tension of the solution becomes equal to the Coulomb repulsion.At this point,the solution at the tip of a needle forms a cone shape,called the“Taylor cone”[27]. If the electricfield intensity is larger than a balance point,small droplets are sprayed spontaneously from the tip of the Taylor cone into a counter electrode.As discussed widely in the literature,the formation of a Taylor cone is essential in electrospray and electro-spinning for the production of droplets andfibers.The formation of a Taylor cone generally requires certain operational conditions, such as particular feed rate,surface tension,conductivity,voltage, and so on[28–30].In the present study,a sodium alginate aque-ous solution was electrosprayed from a needle with a high voltage and the formation of a Taylor cone was also confirmed(inset of Fig.1),similar to previous reports[7,31,32].The charged droplets of the sodium alginate solution contacted the chitosan solution on the counter electrode and each immediately formed a polyelec-trolyte complex by an electrostatic interaction at the interface of the alginate solution/chitosan solution,resulting in a microcapsule. The alginate/chitosan microcapsules obtained by electrospray are shown in Fig.2a.The diameters of the microcapsules were from115 to145m,with a relatively narrow diameter distribution(Fig.2b), giving a coefficient of variation(CV)of only8.0%.These results indicate that the electrospray technique continuously produced aqueous droplets of a uniform size and that the droplets sprayed into the atmosphere reached the chitosan solution without expe-riencing droplet coalescence in the atmosphere during theirflight, probably due to the repulsion between charged droplets[33].We then investigated the effects of the operational conditions on the diameter of alginate/chitosan microcapsules.When varying one of the parameters,the other parameters were kept the same as those in Fig.2.Fig.3a shows the effect of feed rate on the diameter of the micro-capsules.The diameter of the microcapsules decreased from130 to80m with decreasing feed rate.The standard deviation also decreased with the diameter of microcapsules.Next,the effect of the working voltage on the diameter of microcapsules was stud-ied(Fig.3b).In the absence of a working voltage,the diameter of the microcapsules was around2.0mm,which agreed with previous reports[15,16].The electrostatic repulsions at the liquid level on the Taylor cone became stronger as the voltage increased,so that the droplets became smaller.As a result,the diameter of the result-ing microcapsules decreased with increasing voltage.As shownin Fig.2.(a)Microscope image and(b)size distribution of alginate/chitosan micro-capsules prepared by electrospray.Operating conditions:1.5wt%sodium alginate (0.2mL/h),0.5wt%chitosan,voltage22.5kV,needle-to-collector distance5cm,and inner/outer needle diameters130/310m.The scale bar represents100m.Fig.3c,we examined the effect of the needle-to-collector distance on the diameter of microcapsules.The diameter decreased with decreasing distance.This was probably due to the same reasons as given above for the effect of the working voltage:the shorter distance,the stronger the intensity of the electricfield.Fig.3d shows the effect of the inner diameter of the needle(130, 190,330and900m).The diameter of microcapsules increased linearly as the inner diameter of the needle increased from130to 330m.When a needle with an inner diameter of900m was used,the microcapsules were polydisperse(CV=39.3%).This is because the Taylor cone became unstable when large needle diam-eters were used.Interestingly,the diameters of the microcapsules were nearly the same as or smaller than the inner diameters of needles,even though there was negligible evaporation of water.In other meth-ods(e.g.,membrane emulsification,coacervation and microfluidic techniques),it was not possible to produce microcapsules smaller than the nozzle or pore diameter.This feature of the electrospray technique suggests the production of microcapsules and micro-spheres smaller than the limitations of microfabrication techniques is possible and this would be a great advantage in controlling the diameter of microcapsules and microspheres.The effect of the sodium alginate concentration(1.0,1.5,2.0wt%) was studied(Fig.3e).The pH of each sodium alginate solution was around7.5.At a sodium alginate concentration of1.0wt%, the mean diameter of microcapsules was95m with a CV of18%,Y.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects370 (2010) 28–3431Fig.3.Effects of electrospray operating conditions on the diameter of microcapsules.(a)Feed rate,(b)working voltage,(c)needle-to-collector distance,(d)inner diameter of needle,(e)concentration of sodium alginate,(f)concentration of chitosan.Typical operating conditions were1.5wt%sodium alginate(0.2mL/h),0.5wt%chitosan,working voltage22.5kV and needle-to-collector distance5cm.Needle inner/outer diameters were130/310m.while the CV at1.5and2.0wt%were only9.9and9.3%,respec-tively.At0.5wt%,no spherical microcapsules were observed.When the concentration of sodium alginate is low,an alginate/chitosan complex would be formed but such a complex might be soluble in the chitosan solution because of the excess amount of polyca-tions present.The concentration of the chitosan solution,which received the droplets of alginate solution,also affected the diam-eters of microcapsules(Fig.3f).When the chitosan concentration was set at1.0,1.5and2.0wt%(pH3.9,4.1and4.3),the diame-ters of microcapsules were relatively small and there were many non-spherical microcapsules(inset of Fig.3f).The spherical micro-capsules were formed at0.5wt%chitosan solution(pH3.6).It can be presumed that the shape of microcapsules depends on the forma-tion rate and mechanical strength of the polyelectrolyte complex [34,35].There is room for further investigation on the mechanism of the microcapsule formation considering the formation rate and the mechanical strength of the polyelectrolyte complex.Not only the combination of alginate/chitosan but various other combinations of polyelectrolytes were also employed to pre-pare polyelectrolyte microcapsules.The combination of PSS/PAH (PSS sprayed into PAH),PAH/PSS,chitosan/PSS and PDDA/PSS were investigated(Fig.4).Although the combination of PSS/PAH (Fig.4d)also produced observable microcapsules,they had insuf-ficient mechanical strength for manipulation by pipetting.While a polyanion solution was sprayed into a polycation solution in the above investigations,we also sprayed a polycationic aque-32Y.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects370 (2010) 28–34Fig.4.Microscope images of microcapsules produced from (a)PAH/PSS,(b)PDDA/PSS,(c)chitosan/PSS,(d)PSS/PAH.The concentrations of PAH,PSS and PDDA solutions were 10wt%and the concentration of chitosan solution was 9wt%,the feed rate was 0.20mL/h,the working voltage was 24.0kV and the needle-to-collector distance was 5.0cm.The inner/outer diameters of the needle were 330/630m.The scale bars represent 100m.ous solution into a polyanionic aqueous solution.As shown in Fig.4a,PAH/PSS microcapsules were successfully prepared.The mean diameter of the resultant microcapsules was 210m and the CV was 22%.Chitosan/PSS microcapsules (Fig.4c)were also prepared but were polydispersed.One of the reasons for the poly-dispersed chitosan/PSS microcapsules may have been that the lack of a balance of forces leading to an unstable Taylor cone,caused by high viscosity of the chitosan solution.The combination of PDDA/PSS (Fig.4b)produced microcapsules but their mechani-cal strength was as low as that of the PSS/PAH microcapsules (Fig.4d).Finally,we investigated the encapsulation of protein,dextran and cells in the polyelectrolyte microcapsules by electrospray.When protein,dextran and cells are encapsulated,the encapsula-tion efficiency and the residual activity are of considerable practical importance.In this electrospray technique,no organic solvent,heat or vacuum for drying are required.Therefore,it is expected that this method would be suitable for encapsulation of physi-ologically active substances and cells.In this study,fluorescent microspheres,albumin–FITC,TRITC–dextran and yeast cells were used as core substrates and a sodium alginate solution containing each core substrate was electrosprayed into a chitosan solution.Taking into account the capsule morphology,the productivity and stability of Taylor cone,the following production parameters were chosen to prepare alginate/chitosan microcapsules encapsulating the substrates;the concentrations of sodium alginate and chi-tosan were 1.5,0.5wt%,respectively,the feed rate of the sodium alginate solution was set at 0.20mL/h,the working voltage was 22.5kV,the distance from the needle to the collector was 5.0cm and the inner/outer diameters of a stainless steel needle were 130/310m.To approximate the encapsulation efficiency of yeast cells,we measured that of fluorescent microspheres because the size of fluorescent microspheres and yeast cells are nearly equal (a couple of micrometers).The CLSM images of the microcap-sules revealed that these fluorescent substances (the fluorescent microspheres and albumin–FITC)were uniformly spread over the whole microcapsules composed of alginate/chitosan,meaning that these substances were successfully encapsulated in the monodis-persed microcapsules (Fig.5).The encapsulation efficiency of microspheres and albumin was >99%.It should be noted that the diameters of the microcapsules and the size distribution were not affected by the presence of core substrates.These results demon-strate that the electrospray technique can encapsulate micro-and nano-sized materials in polyelectrolyte microcapsules with a high encapsulation efficiency.The retention of TRITC–dextran with different molecular weights in microcapsules was investigated.The retention ratios of all types of TRITC–dextran (Mw =4400,65,000–76,000and 155,000)in sodium alginate/chitosan microcapsules were >99%at 24h after dispersing microcapsules in an acetate buffer.Pre-vious studies reported that the ultrathin films of polyelectrolytes prepared by the layer-by-layer method displayed similarly high rejection properties to nanofiltration membranes [36–38].The high retention properties of the microcapsules in the present study were therefore reasonable.Fig.6shows phase-contrast microscope images of microcap-sules encapsulating yeast in a YPD medium at different periods after microcapsule preparation.As is evident from these images,yeast cells were also successfully encapsulated in the alginate/chitosan microcapsules and they grew inside the microcapsules over time.The image at 0h and the results from fluorescent microspheres allow us to speculate that most of cells sprayed were encapsulated within the microcapsules.The growth of yeast in a microcap-sule indicates sufficient penetration of low-molecular materials (glucose,peptone,etc.)from the culture medium through the microcapsule shell consisting of the alginate/chitosan complex.Even after 48h,microcapsules containing yeast cells did not tear,which indicates considerable mechanical strength of the algi-nate/chitosan microcapsules.These results demonstrate that the electrospray technique achieves the encapsulation of a physiolog-ically active substrate into polyelectrolyte microcapsules without any critical damage to the substrate.In summary,we prepared microcapsules based on cationic and anionic polyelectrolytes by the electrospray technique.The electrospray technique produced monodispersed alginate/chitosan microcapsules,whose size was controlled by varying the operatingY.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects 370 (2010) 28–3433Fig.5.CLSM microscope images of alginate/chitosan microcapsules containing (a)and (b)fluorescent microspheres,(c)albumin–FITC,(d)TRITC–dextran (Mw =155,000).Operating conditions were the same as those in Fig.2.The scale bars represent 100m.conditions.This technique can utilize various types of natural and synthetic polyelectrolytes,and also encapsulate various substrates (biomacromolecules,microspheres and living cells)in the micro-capsules with high encapsulation efficiency and without critical damage to the substrates.Due to the simplicity of the electro-spray setup and the attractive properties discussed above,the electrospray technique is expected to be a practical method for the industrial production ofmicrocapsules.Fig.6.Phase-contrast images of yeast-encapsulated microcapsules in YPD media at different periods after the preparation of microcapsules.(a)0h,(b)8h,(c)16h,(d)24h.Operating conditions were the same as those in Fig.2.The scale bars represent 100m.34Y.Fukui et al./Colloids and Surfaces A:Physicochem.Eng.Aspects370 (2010) 28–34AcknowledgmentsWe thank Professor M.Kotaki at Kyoto Institute of Technology for the technical support.This work was supportedfinancially by Special Coordination Funds for Promoting Science and Technol-ogy,Creation of Innovation Centers for Advanced Interdisciplinary Research Areas(Innovative 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