Assembly of multilayer PSS/PAH membrane on coherent alginate/PLO microcapsule for long-term graft...

Assembly of multilayer PSS/PAH membrane on coherentalginate/PLO microcapsule for long-term grafttransplantation

Andy Leung, Matt Trau, Lars Keld NielsenAustralian Institute of Bioengineering and Nanotechnology, University of Queensland, Brisbane,Queensland 4072, Australia

Received 24 April 2007; revised 9 July 2007; accepted 9 November 2007Published online 19 February 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31891

Abstract: Conventional alginate/poly-L-ornithine (AP)membranes used to immunoisolate foreign tissue transplantsfail in long-term transplantations of immortal cell lines. Wehave developed a novel layer-by-layer (LbL)membrane usingpolystyrene sulfonate and polyallylamine hydrochloride(PSS/PAH) on top of the coherent AP membrane. Assemblyof the LbL membrane was followed by electrophoresis, andthe surface morphologies and structure were characterizedand examined by cryo-scanning electron microscope and

transmission electron microscopy. Unlike the standard APmembrane, the LbL membrane withstood the internal pres-sure generated by continuous cell proliferation of microen-capsulated HEK-293 andMin-6 cells. The newmembrane didnot affect insulin secretion or diffusion by Min-6 cells.� 2008Wiley Periodicals, Inc. J BiomedMater Res 88A: 226–237, 2009

Key words: microencapsulation; continuous cell line; integ-rity; layer-by-layer membrane; insulin secretion

INTRODUCTION

Microencapsulation is used to contain andimmuno-protect cells used in cell-based drug ther-apy. Cell-based drug therapy enables direct, continu-ous supply of drugs in difficult to access sites andhas been studied in transplantation models of Par-kinson’s disease,1,2 Alzheimer’s disease,3 chronicpain syndrome4,5 as well as for localized predrugactivation in pancreatic cancer treatment.6 Cell-baseddrug therapy also enables physiologically responsivedrug delivery, and the treatment of type I diabetesby transplantation of insulin-producing cells hasbeen the major focal interest of this approach andwas shown to be successful in reversing diabetes inan animal model.7 The most common biocompatiblemembrane used in cell microencapsulation is the oneprepared from alginate and poly-L-lysine (PLL).

We have previously demonstrated a mild processto produce highly organized and functional microtis-sue from various cell lines employing the hangingdrop method.8 The method can easily transform

immortalized cell lines with desired biochemicalfunctionality into micron-sized cell spheroids, whichcan then be encapsulated with a coherent alginate/poly-L-ornithine microcapsule by our emulsion pro-cess to produce a readily transplantable cell-baseddrug delivery device.9 This thin alginate coherentmicrocapsule conforms closely to the spheroid’s sur-face, which virtually eliminates any dead space inthe microcapsule and hence minimizing the trans-plant volume.

Mechanical integrity and long-term stability ofalginate microcapsules

The durability of the microcapsule device used fortransplantation is largely determined by the mechan-ical integrity and chemical stability of the membrane.Mechanical fragility is one of the major drawbacksto the development of microcapsules. Studies haveshown that the biocompatibility of the conventionalalginate-based microcapsule is strongly dependenton the binding efficiency of the polyelectrolytes ofthe membrane.10 With a weak polyelectrolyte attach-ment, the membrane detaches quickly and fre-quently after implantation, which leads to the rapiddestruction of the transplanted materials by the hostimmune system. Various strategies for enhancing thestructural integrity and long-term stability of the al-

Correspondence to: L. K. Nielsen; e-mail: [email protected] grant sponsor: Australian Research Council;

contract grant numbers: A29937058, FF0455861

� 2008 Wiley Periodicals, Inc.

ginate microcapsules have been studied in the past,including the use of different multivalent cations toform the core alginate hydrogel,11,12 different polyca-tions on the alginate microcapsules,13,14 and employ-ing enzyme reactions to modify the mannuronic acidand guluronic acid ratio of alginate to enhance thebinding of PLL onto the alginate microcapsules.15

Layer-by-layer membrane

There have been many studies on the manufactureof polyelectrolyte multilayer microcapsules with theprocess of layer-by-layer (LbL) adsorption of oppo-sitely charged polyelectrolytes onto charged colloidalparticles with subsequent removal of the templatecore.16–19 Thin membranes prepared by this LbLadsorption technology using a variety of materials,including synthetic polyelectrolytes, biopolymers,and inorganic particles, have been studied for appli-cations in drug delivery, catalysis, and other biotech-nology with desired properties.18,20–22 Capsules witha specific control for the release of encapsulated mol-ecules had been developed with multiple polyelec-trolyte layers and light-responsive nanoparticles inthe preparation of the polyelectrolyte membrane.23

This technology has been applied for assemblingmultilayer membranes on the surface of colloidalparticles, and for the production of micron-sizedstable hollow polyelectrolyte capsules by subsequentdecomposition of the colloidal core. One of the sig-nificant properties is the selective permeability ofthis polyelectrolyte membrane. Previous studieshave shown that a molecular weight cut-off(MWCO) of 5000 Da can be achieved by this technol-ogy, through which small molecules and ions canpromptly diffuse.24,25

LbL membranes have been used to tailor the func-tionality of conventional microcapsules to achievethe objectives of biocompatibility, molecular sizeexclusion, and mechanical stability.26 When used onislets encapsulated by the conventional alginatedroplet method, LbL membranes were shown to notaffect insulin secretion, and the resultant device wasstable for 4 weeks in vivo.20

One of the most investigated polyelectrolyte sys-tems is based on polystyrene sulfonate and polyallyl-amine hydrochloride (PSS/PAH), which has beensuccessfully fabricated into stable and porous hollowmicrocapsules by subsequent core disintegration af-ter encapsulation.19,27–30 This study deals with theusage of PSS/PAH as templates for LbL multilayerassembling on alginate-based microcapsules to facili-tate structural integrity and stability for transplanta-tion of an immortalized cell line as a drug deliverysystem. This multilayer PSS/PAH membrane hasbeen demonstrated to be stable in various chemical

environments19,27,31 and represents a convenientapproach to enhance the structural integrity of algi-nate/PLO (AP) microcapsules. The core of calciumalginate microcapsules is an excellent platform tosupport the encapsulated cell spheroids; however, italone is rather fragile, and often a supporting poly-mer, such as PLL or poly-L-ornithine, is incorporatedon the microcapsule surface to enhance the struc-tural integrity. AP membranes can contain tissuesthat have minimum cellular growth and retain theirvolume, such as the Islets of Langerhans. However,for cell spheroids prepared from modified immortal-ized cell lines, the stress and pressure generatedfrom the cell proliferation inside the microcapsulesis enough to rupture and disintegrate the encapsulat-ing alginate membrane. Losing the protective mem-brane by any means will instantaneously activate thehost immune system and subsequently destroy allexposed foreign transplanted materials. As proof ofconcept for the proposed device, this study focusedon the LbL multilayer membrane composed of fourlayers of alternating PSS and PAH assembled on acoherent AP microcapsule. The capability of the pro-posed LbL multilayer membrane in the applicationof transplanting the microencapsulated insulin-pro-ducing cell, MIN-6, was investigated.

MATERIALS

Dulbecco’s modified Eagle’s medium (DMEM) withhigh (25 mM, 10313-021) and low (5 mM, 11885-084) glu-cose, fetal bovine serum (FBS), L-glutamine (25030-081),and penicillin/streptomycin/fungicide (PSF) were ob-tained from Gibco. Medium viscosity alginate (A2033),Ficoll 400 (F2637), polyethylene glycol (P6667), poly-L-orni-thine hydrochloride (P2533), polystyrene sulfonate(243051), and PAH (283223) were purchased from Sigma.A Sensitive Rat Insulin Radioimmunoassay (RIA) kit (SRI-13K) was obtained from Linco Research.

METHODS

Cell spheroids preparation

HEK 293 cell spheroids were prepared in DMEM cul-ture medium, supplemented with 10% FBS, by the hangingdrop method8 using Nunc MicroWell Minitrays in an incu-bator equilibrated with 5% CO2 and 95% air at 378C. Sphe-roids were grown to reach a diameter in the range 200–250 lm and typically were spherical or ovoid in shape. Toassess the maximum size of the spheroids, cell spheroidswere cultured at different initial seeding concentrations of250, 1250, and 2500 cells per well (20 lL) on minitrays forextended periods of time. After 7 days, the cell spheroidswere transferred from the minitrays to a 96-well plate with100 lL of fresh culture medium. At day 15, the medium

MULTILAYER PSS/PAH MEMBRANE FOR LONG-TERM GRAFT TRANSPLANTATION 227

Journal of Biomedical Materials Research Part A

was replaced with fresh culture medium. The average di-ameter of each cell spheroid was measured using light mi-croscopy and was recorded at subsequent time intervals.

AP microcapsules

Alginate coats were formed on the cell spheroidsthrough an adaptation of the aqueous emulsion methoddeveloped by Calafiore and coworkers.8,14 0.2% (w/v)sodium alginate and 11% (w/v) PEG were dispersed in10 mL of salt solution containing 5 mM KCl/145 mMNaCl as osmolytes to adjust the aqueous environment tosupport the cells. Separately, a 40% (w/v) Ficoll was pre-pared in the same salt solution. Both the sodium alginate/PEG mixture and Ficoll were sterilized by gamma-radia-tion at 25 kGrays prior to dissolving in the salt solution.

The standard emulsion was composed of 1 mL of the al-ginate/PEG solution mixed with 150 lL of Ficoll solutionand vortexed for 10 s to generate a uniform distribution ofemulsion droplets. It was then rotated laterally on a com-puter controlled stepper motor rotator at a speed of 7 rpmfor 20 min to obtain the desired size of the Ficoll dispersedphase. HEK 293 cell spheroids, which had been harvestedafter culturing as described earlier for 4 days and hadreached a diameter of around 200 lm, were washed withTBS and transferred into the emulsion solution. The solu-tion was then mixed gently using a pipette to disperse thecell spheroids and prevent clusters of cell spheroids thathad encapsulated together.

The emulsion mixture was then rotated laterally on acomputer controlled stepper motor rotator at a speed of1 rpm for 10 min. Afterwards, the emulsion mixture wasbriefly hand-shaken to break up large Ficoll clumps andwas then gently transferred to 10 mL of 0.1M CaCl2 solu-tion to facilitate the calcium crosslinking of the alginatemembrane. The microencapsulated cell spheroids werewashed and resuspended in 0.1M CaCl2 solution.

Subsequently, 0.1% (w/v) PLO in 0.15M NaCl wasadded to the microencapsulated cell spheroids at a 1:5 ra-tio, followed by vortexing and then mixing on a rotator for15 min to form the AP microcapsules. The AP microencap-sulated cell spheroids were washed with PBS to removeexcess PLO and cultured in the DMEM-based culture me-dium equilibrated with 5% CO2 and 95% air at 378C.

LbL polyelectrolyte assembly on AP microcapsules

Assembly of the proposed LbL polyelectrolyte mem-brane microcapsules used an alternating adsorption pro-cess. The AP microencapsulated HEK 293 cell spheroidswere generated using the methods described earlier andwere suspended in 0.1M NaCl solution.

Then, 0.1 mL of 1 mg/mL PSS in 0.1M NaCl was addedto 1 mL of a suspension of the AP microcapsules. The mix-ture was vortexed and mixed on rotator for 15 min. Thesuspension was centrifuged in a minicentrifuge, the super-natant was removed and 1 mL of 0.1M NaCl was added tothe suspension. This process was repeated twice to removethe unbound polyelectrolytes. 0.1 mL of 1 mg/mL of PAHin 0.1M NaCl was added to the microcapsule suspension

that was again mixed on a rotator for 15 min to form thePSS/PAH multilayer by adsorption. Next, three washingsteps were carried out as described earlier to remove non-adsorbed molecules. This process of adding the PAH solu-tion and then washing was repeated until four layers ofPSS/PAH multilayer membranes were assembled on themicrocapsules. The PSS/PAH multilayer-microencapsu-lated HEK 293 cell spheroids were then cultured in aDMEM based culture medium equilibrated with 5% CO2

and 95% air at 378C.

Zeta potential measurements

Measurements of the zeta potential on the surface layerson the alginate beads were performed on a Malvern Zeta-sizer. Alginate beads made from the emulsion methodwere vortexed prior to calcium crosslinking to producesmall alginate beads suited for the zetasizer experiment.Alginate beads of 1–2 lm in diameter were obtained byrepeated centrifugation to remove the unwanted largerones. The selected micron-sized alginate beads were thenwashed thoroughly in MilliQ water before zeta-potentialswere obtained. The polyelectrolytes of PLO, PSS and PAHwere sequentially adsorbed onto the core alginate beadsthat were washed extensively by MilliQ water prior toobtaining each of their zeta-potentials from the MalvernZetasizer.

Integrity study of the microcapsules

The long-term integrity of the different microcapsules,the original AP membrane and the multipolyelectrolytemembrane, were assessed by examining the cell spheroidsimmediately after coating and at subsequent time intervalsunder light microscopy. To test the integrity of the APmicrocapsules in conditions similar to that in the humanbody, the AP microencapsulated HEK 293 cell spheroidswere cultured in DMEM-based culture medium equili-brated with low oxygen at 378C. The culture medium waschanged at day 7 and day 13.

To test the effectiveness and structural integrity of theproposed PSS/PAH multilayer-microcapsules, HEK 293cell spheroids microencapsulated with the PSS/PAH mul-tilayer membrane were cultured and examined under thestandard laboratory culturing method for 7 days.

Cryo-scanning electron microscope

To examine the surface morphologies and properties ofthe original alginate hydrogel surface, the AP membraneand the PSS/PAH multilayer microcapsules, cryo-scanningelectron microscope (Cryo-SEM) was employed using aJEOL 6300F with OXFORD CT 1500 Cryo-Preparation Sys-tem. Firstly, samples of calcium alginate beads, AP mem-brane microcapsules, and the PSS/PAH multilayer micro-capsules were prepared using the above procedures, omit-ting the step where the cell spheroids were added.

The samples were individually examined by transferringa concentrated sample on to a sample stub covered by a

228 LEUNG, TRAU, AND NIELSEN


small piece of filter paper glued to the top to absorb theexcess water from the bead samples. The sample was cryo-fixed by plunging the sample into subcooled nitrogenslush close to the freezing point of nitrogen at 22108C.This liquid–solid nitrogen slush is useful for quench freez-ing small specimens because it does not boil. It was pre-pared by pumping liquid nitrogen into a partial vacuumwhich then cools by evaporation to the melting point ofnitrogen (22108C).

Sublimation (etching)

Even with the use of the filter paper, there was still re-sidual water and condensed water vapor covering thesample’s surface which obscured the fine structural detailof the polymer bead sample. An etching process, whichraised the temperature of the sample in the SEM Cryo-Stage to around at 2858C, was employed to remove thesurface water by sublimation. The ice sublimation wasmonitored by directly viewing the sample under the elec-tron beam. After 30 min of etching, the sample was rap-idly cooled to the working temperature of the Cryo-SEMat around 21608C. Cryo-SEM images of the sample’s sur-face were obtained using the JEOL 6300F instrument oper-ating at an acceleration voltage of 5 keV.

Transmission electron microscopy

Alginate beads, prepared with different polyelectrolytesurface modifications, were washed with MilliQ waterbefore fixation for transmission electron microscopy(TEM). Centrifuged concentrated alginate beads weremixed with 2% agarose in 100-lm planchettes, to providesupport for the bead after dehydration, and frozen in aLeica EMPACT 2 high-pressure freezer. The samples werecryosubstituted in acetone containing 2% osmium tetroxideand 0.5% uranyl acetate at 2908C for 40 h. They were thenprogressively warmed from 2908C to 208C over 8 h in aLeica automatic freeze substitution unit. After beingwashed in acetone, they were embedded in Epon resin.Ultrathin sections of the alginate bead samples (thicknessof 60 nm) were cut on a Leica Ultracut T ultramicrotomeand stained with 5% uranyl acetate in 50% methanol andReynolds lead citrate. Samples were viewed in a JEOL1010 TEM at 80 kV.

Insulin secretion study

Insulin secretion was determined using a static incuba-tion method in standard culturing conditions with 5% CO2

at 378C. MIN-6 cells were cultured in high glucose DMEM(25 mmol/L glucose) supplemented with 10% FBS, 200mM of L-Glutamine, PSF, and 0.5% of 2-mercaptoethanol.The MIN-6 cell line acquired from the Garvan Institute ofMedical Research was at passage 20 when received andwas used to prepare cell spheroids at passages 23–25. Thecell spheroids were cultivated using the hanging-dropmethod in Nunc MicroWell Minitrays under standard cul-turing conditions until they reached a diameter of around

200 lm. MIN-6 cell spheroids were then encapsulated withthe AP microcapsules and the PSS/PAH multilayer mem-brane by using the emulsion method and LbL process,respectively, following the procedures described earlier. Abatch of unencapsulated cell spheroids was kept as a nega-tive control.

Following the AP and LbL multilayer microencapsula-tion process, the microencapsulated MIN-6 cell spheroids,as well as the uncoated control, were cultured in high glu-cose DMEM medium overnight at 378C for recovery. Allsamples were washed with phosphate-buffered saline andpreincubated for 1 h in low glucose (5 mmol/L glucose)DMEM medium supplemented with 10% FBS at 378C.Then they were washed and incubated individually in200 lL of high glucose DMEM medium (with no supple-ment other than 200 mM of L-Glutamine) in a 96-wellplate at 378C for 15 min, 1 h, 2 h, and overnight (�17 h).The supernatant of each sample was collected at the desig-nated time interval, pipette-mixed, and stored in thefreezer immediately.

A Sensitive Rat Insulin RIA kit was obtained from BDBiosciences and the radioimmunoassay was performed byIDEXX Laboratories (Brisbane, Australia).

RESULTS AND DISCUSSIONS

Cell spheroids size

For optimal biomaterial transfer and effectivenessin implantation of such a device in the body, themicrocapsule needs to be in the range of 200–300 lmto facilitate high surface-volume ratio and conven-ient delivery by injection.

Figure 1 shows that the cell spheroids preparedfrom the HEK 293 cell line have the ability to growcontinuously as long as fresh culture medium ispresent. The spheroids were cultured at three differ-ent initial seeding densities. Those with 1250 cells

Figure 1. Growth rate of HEK 293 cell spheroids preparedfrom different initial seeding concentration of 2500 cells(l), 1250 (*) and 250 (!) per well by the hanging dropmethod. Error bars represent standard deviation (n 5 10).



and 2500 cells per well both reached an average di-ameter of 250 lm after 2 days of culture in standardconditions and remained at that size, whereas thecell spheroids with 250 cells per well developedslowly, reaching a average diameter of around150 lm on day 2 and slowly grew to 250 lm after7 days. Initially, it appeared that the cell spheroidshad attained their maximum size, with the prolifera-tion and death rate reaching a steady state.

However, after the cell spheroids were transferredto a greater volume of fresh DMEM culture mediumin a 96-well plate, all three batches of cell spheroidsbegan to grow again, reaching a diameter of around500 lm at day 15 and growing further to 650–700lm after renewal of DMEM culture medium. Insteadof having the dead cells collapse and disintegrateinside the core of the cell spheroids, the new cellscontinued to proliferate at the surface region andgrew outward from the core. Thus, the limiting fac-tor for the size of the cell spheroids is the availabilityof glucose, amino acids, and other nutrient substan-ces in the culture medium.

Mechanical failure of the AP microcapsules

The problem with cell spheroids prepared fromimmortalized cell lines is the rapid rupturing of themicrocapsule due to continuous proliferation of thecellular material. This is a major limitation in usingimmortalized cell line as a drug delivery platform,because the microcapsules self-destruct and triggerthe immune system to consume the implanted mate-rials too quickly for any practical application.

The durability of the conventional AP microcap-sules made from the emulsion technique that encap-sulates cell spheroids prepared from immortalizedcell lines under standard and low oxygen cultureconditions was examined by light microscopy over aperiod of 2 weeks. Microcapsules with a visuallyintact membrane covering the whole cell spheroid[Fig. 2(a)] were considered as having a functionalmembrane, whereas the microcapsules with cellularmaterials rupturing from the membrane [Fig. 2(b)] orwith a visually torn membrane [Fig. 2(c)] were con-sidered failures.

The results are summarized in Figure 3. HEK 293cell spheroids encapsulated with the AP microcap-sules were shown to have ruptured and torn APmembrane with the cellular material protrudingsoon after being encapsulated. After 6 days in stand-ard culturing conditions, only 50% of the AP micro-capsules were intact and functional. The failure of theAP microcapsules continued at a similar rate and onlyabout 5% of the total were considered to have validintact membranes after 13 days in standard culture.To simulate in vivo conditions, AP microcapsules

with spheroids prepared from the HEK 293 cell linewere cultured in low oxygen, which in theoryshould have slowed down cell proliferation in thecell spheroids and hence have reduced the expan-sion force and internal pressure on the microcapsulemembrane. Yet the results only varied slightly fromthe viability of the AP microcapsules under standardculturing conditions. Again, about 50% of the APmicrocapsule had failed after 6 days. The rate ofmicrocapsule destruction was slower than thoseunder standard conditions, with about 20% of thetotal microcapsules deemed to be functional after13 days.

LbL PSS/PAH polyelectrolyte membrane

The AP membrane is clearly not suitable formicroencapsulating an immortalized cell line. ThePSS/PAH LbL membrane has previously beenemployed for building permeable and stable mem-branes for other hollow capsule fabrications.

The LbL assembly of polyelectrolytes on the APmicrocapsules is a simple process to strengthen themembrane with no harmful reaction to the encapsu-lated cells. To determine the material used for build-ing this LbL membrane, the essential factor is thatthe adsorbed layer has to be oppositely charged tothe existing membrane’s surface. It has been shownthat the PSS/PAH LbL membrane can be assembledon both positively and negatively charged colloidalparticles and can only be dissociated at a tempera-ture of 808C.27

Electrophoresis was performed to follow the LbLadsorption of the PSS/PAH multilayers on the APmicrocapsules. The solutions of polyelectrolytes weresequentially added to the suspension of the APmicrocapsules. Washing with saline solution toremove unbound materials was performed after eachadsorption stage. To avoid errors due to rapid sedi-mentation during measurements, small alginatemicrocapsules were prepared for this experiment.Figure 4 shows the zeta-potential measurement ofthe core alginate, the initial PLO layer, and the sub-sequent PSS/PAH multilayers, starting from about240 mV corresponding to the core calcium alginatehydrogel. The zeta-potential alternates according tothe last adsorbed outer layer. The surface chargechanged to positive zeta-potential values after theadsorption of PLO and PAH, and the surface chargechanged to a negative value with the adsorption ofPSS. The observed behavior of the alteration of thesurface charge with addition of oppositely chargedpolyelectrolytes is very similar to that known forpolyelectrolyte adsorption.22



Figure 2. Bright field optical microscopy images of (a) functional complete coherent AP membrane around HEK 293 cellspheroids, (b) failed AP microcapsule with cellular materials rupturing from the membrane, and (c) totally destroyed APmembrane with HEK 293 cell spheroids growing on the microwell bottom. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]



Cryo-SEM and TEM study of the surfacemorphology of the LbL PSS/PAH multilayermembrane

Surface analysis of conventional calcium alginateand AP microcapsules, as well as the proposed LbLPSS/PAH membrane, were studied using Cryo-SEMand TEM imaging.

A typical Cryo-SEM image of the core alginatemicrocapsule is shown in Figure 5(a). The microcap-sule had completely collapsed, losing the sphericalshape. The thin columns on the surface are consid-ered to be the collapsed fine structure of the alginatematrix. Because the alginate hydrogel consistsmostly of water, the fine structure of the calciumcrosslinked alginate matrix is not strong enough to

support its matrix structure in a vacuum, and hencethe polymers of the outer surface of the hydrogelcollapse onto each other to form large gaps and flat-tened areas on the surface.

A typical Cryo-SEM image of the AP microcapsuleis shown in Figure 5(b). The surface had a ratherlarge porous morphology. It can be observed that

Figure 3. Viability of the conventional AP membraneencapsulating cell spheroids prepared with the HEK 293cell line under standard culturing conditions (*) and lowoxygen (!) conditions.

Figure 4. Zeta potential data of consecutive PSS/PAHmultilayers incorporated on uncoated alginate microcap-sules via the LbL adsorption method as a function of layernumbers. The odd layer numbers correspond to PLO andPAH adsorption and the even layer numbers to PSSadsorption.

Figure 5. Cryo-SEM images of (a) core alginate microcap-sule, (b) core alginate/PLO microcapsule, and (c) LbLPSS/PAH multilayer membrane.



the microcapsules had considerable shrinkage andstructural collapse on the edge, yet the overall spher-ical structure of the microcapsules can still be identi-fied. It is known that sublimation can result in alevel of freeze-drying and associated shrinkage ofspecimen tissue. Although the removal of water canlead to shrinking artifacts, this must be traded offagainst the fact that some structures can only beseen when others have collapsed.32

The gap on the surface cannot be the actualporous matrix structure of the alginate hydrogelbecause the visible gap is too big to produce therecorded MWCO size of the microcapsules.

The typical Cryo-SEM image of the LbL PSS/PAHmembrane microcapsule is shown in Figure 5(c). Themicrocapsules have retained the overall sphericalstructure with no shrinkage, and a much clearer anddistinct homogenous surface can be observed. TheLbL membrane had the smallest visible pore size com-pared with the core alginate and AP membrane. Theextra PSS and PAH polymers assembled on the sur-face of the microcapsules provided the supplementarystructural support to maintain and uphold the fineoriginal matrix on the surface of the membrane.

The effect of reduction on the degree of collapseand shrinkage of the microcapsules during surfaceetching, as well as the finer details of the surface ma-trix structure of the microcapsules observed in theCryo-SEM images, have no direct relationship withthe membrane mechanical strength in their hydratedstates. However, it does demonstrate that additionalpolyelectrolyte materials have been assembled onthe AP microcapsules with the LbL methods andthat there is a general difference in terms of mor-phology between the pure alginate, the AP, and theLbL PSS/PAH membrane.

The TEM images of the uncoated alginate, AP,and LbL PSS/PAH membranes (two different LbLmembranes: one with 4000 kDa PSS and the otherwith 70 kDa PSS) are illustrated in Figure 6(a–d).Because the microcapsules had been fixed in theirhydrated states, TEM can generate cross-sectionalimages of the membranes close to their originalforms in solution.

The pure alginate microcapsules have a rough andundefined surface. Washing with MilliQ water priorto treatment may cause the coarse surface, whichdissolves and slightly destabilizes the alginate ma-trix. The background has a similar texture to the al-ginate core because the microcapsules are suspendedin agarose to provide support after dehydration. TheAP membrane shows a more defined and sharpersurface, with a visible AP membrane on the surfaceof the alginate matrix. The LbL PSS/PAH mem-brane, prepared from PSS with the shorter MW of70 kDa, shows a thick, consistent layer on the surfaceregion [Fig. 6(d)]. The LbL membrane made from a

larger MW of 4000 kDa PSS shows a bulky, irregularmembrane on the surface of the alginate microcap-sules [Fig. 6(c)]. It is suggested that the observedthicker layer of LbL membrane with the shorter MWPSS is due to assembly of the shorter polyelectrolytesof 70 kDa PSS and 70 kDa PAH into the core algi-nate matrix. On the other hand, the multilayersassembled with the larger MW PSS could not diffuseinto the alginate hydrogel. The TEM images clearlyshow that the LbL adsorption process can solidifythe outer membrane of the alginate-based microcap-sule by assembling addition polyelectrolytes.

Structural integrity of the LbL membrane

As discussed in the previous section, the mechani-cal stability is one of the most important physicalproperties of the microcapsules. It defines the defor-mation and rupture of the microcapsule membraneunder both external and internal loads, which is anessential characteristic in the development of deliv-ery systems for pharmaceutical drugs.

We first compared the mechanical behavior of theLbL membrane with conventional AP microcapsulesusing osmotically induced buckling of cell free cap-sules immersed in a high concentration of high mo-lecular weight polyelectrolyte solution. Some of theAP microcapsules showed some shrinkage and wrin-kles on the surface, whereas the LbL membraneremained smooth (data not shown). However, no de-finitive and quantitative results could be obtained, asnone of the microcapsules showed complete destruc-tion or were significantly deformed from the theiroriginal spherical shape. We attribute the limitedshrinkage to the fact that the core alginate hydrogelof the microcapsule was made from a Ficoll/PEG-based emulsion and retain most of the 40% Ficoll so-lution carried over from the emulsion solution inaddition to the 2% calcium alginate. This limitswater removal caused by high external osmotic pres-sure. A second attempt based on the opposite princi-ple, swelling of the AP and LbL microcapsule inwater, similarly yielded no definitive conclusion,because the degree of swelling was minute and diffi-cult to quantify (data not shown).

To firmly establish the degree of mechanicalimprovement and the feasibility of cellular applica-tion for the proposed LbL membrane, HEK 293spheroids microencapsulated with or without theadditional four layers of PSS/PAH membrane werecompared. Previous experiments had shown that theconventional AP membrane encapsulating the HEK293 cell spheroids had a very short life span (Fig. 3).Therefore, the HEK 293 cell line was again utilizedas the source of internal expansion to measure thestructural integrity of the microcapsules, as well as



to assess the resistance of the LbL PSS/PAH mem-brane to the expansion forces induced by cell prolif-eration. The integrity of the microcapsules wereassessed under light microscopy for a period of7 days and the survival curves of both the conven-tional AP and LbL PSS/PAH membranes are shownin Figure 7. Samples from five batches of encapsula-tion (two AP and three LbL) were compared.

The experimental results showed again that theconventional AP microcapsules began to degradeand disintegrate steadily immediately after beingassembled on the HEK 293 cell spheroids. Afterincubation in standard culturing conditions for7 days, only 30% of the AP microcapsules had a func-tionally complete membrane. In contrast, the fractionof LbL PSS/PAH membrane microcapsules failingover the initial 2–3 days was small and remained atabout 85–90% for the rest of the experiment. Thisresult shows that the LbL PSS/PAH membrane has

a significantly stronger structural integrity than theconventional AP microcapsules (hypergeometric test,p 5 1e 214), and attained sufficient resistance againstrupture from internal pressure caused by cell pro-liferation from the immortalized cell line.

Insulin released from MIN-6 cell spheroids

One of the major concerns of the LbL PSS/PAHmembrane is that the thickened polyelectrolytemicrocapsules could reduce the permeability of themembrane and hence deteriorate the functionality ofthe microcapsules as an adequate transplantation de-vice. The study of the microcapsule viability withthe HEK 293 cell line did not assess the condition ofthe cells encapsulated in the LbL membrane, apartfrom the fact that the encapsulated cells were meta-bolically alive (the medium changed color).

Figure 6. TEM image of (a) alginate hydrogel beads, (b) alginate/PLO membrane, (c) LbL PSS/PAH membrane preparedfrom 4000 kDa PSS and 70 kDa PAH, and (d) LbL PSS/PAH membrane prepared from 70 kDa PSS and 70 kDa PAH.



MIN-6 cell line derived from in vivo immortalizedinsulin-secreting pancreatic beta cells was used tostudy the insulin-releasing capacity and effectivenessof the LbL PSS/PAH microcapsules as a transplanta-tion device. The MIN-6 cell line produces a glucose-induced insulin secretion response in a manner simi-lar to the pancreatic beta cells, which respondquickly to a rise in glucose concentration in the cul-ture medium by releasing stored insulin whilesimultaneously producing more.33

Cell spheroids prepared from the MIN-6 cell linewere encapsulated with the conventional AP or withthe LbL PSS/PAH membrane, with unencapsulatedcell spheroids used as controls to assess the overallefficiency of the two microcapsules. All three sam-ples of encapsulated and uncoated cell spheroidswere rested in standard high glucose level culturemedium overnight for recovery. The cell spheroidswere activated in a low glucose level culture me-dium first, and were then individually cultivated inhigh glucose level culture medium. The insulin con-tent in the culture medium released from eachencapsulated or unencapsulated MIN-6 cell sphe-roids at different time intervals was measured byBD’s Sensitive Rat Insulin RIA kit.

A single MIN-6 cell spheroid was suspended inthe minimal sampling volume of 200 lL required bythe RIA kit. Given the low cell density, the insulincontent measured from all three samples at 15-minand 1-h intervals did not increase over backgroundlevel (Fig. 8). At 2 h, there was a detectable increaseof insulin from the uncoated control, but there wereno significant detectable insulin response from theconventional AP or the LbL PSS/PAH membrane.

After overnight incubation, a distinct accumulatedinsulin release response from all three samples wasobserved. The accumulated insulin released from theunencapsulated control reached an average of0.8 ng/mL, whereas the release from the conven-tional AP and LbL membrane reached an average of0.55 ng/mL and 0.48 ng/mL, respectively. It can beseen that the cell spheroids in both microcapsulesreached only around 60–70% of the total insulinreleased from the unencapsulated control in thesame time interval (t test, p 5 0.02). This differencemay have been caused by the alginate hydrogel corein both types of microcapsules, which acts as an ab-sorbent and trapped a fraction of the insulin releasedfrom the cell spheroids within. On the other hand,there was no significant difference in the insulin con-tent in culture medium from the conventional APand LbL PSS/PAH membrane (t-test, p 5 0.30).

The results demonstrates that the LbL membraneis permeable for glucose and other necessarynutrients to be absorbed by the encapsulated cells,and allow small pharmaceutical drugs, such as insu-lin, to diffuse through the microcapsules membrane.Similar to the previous study on insulin secretionfrom islets through an LbL-modified conventionalneedle drop alginate microcapsules, there is no nega-tive effect on the insulin release from our coherentmembrane encapsulated Min-6 cell line in our study.The microcapsules retained most of the transportproperties after the assembly of the LbL PSS/PAHmembrane. In addition, the encapsulated cells arealive and functional after the LbL membrane hadbeen assembled on the core alginate microcapsulesin a controlled environment. The LbL process ofassembling the PSS/PAH multilayer membrane onthe alginate microcapsules has not altered or dis-

Figure 8. Accumulation of insulin in MIN-6 cell sphe-roids, uncoated and microencapsulated in conventional APand LbL membrane prepared from 70 kDa PSS and 70 kDaPAH. Error bars represent standard deviation (n 5 3).

Figure 7. Survival curves for microcapsules with conven-tional AP membrane and microcapsules with the LbLmembrane prepared from 70 kDa PSS and 70 kDa PAH.Two batches of microcapsules with conventional AP mem-branes (dotted line) were compared with three batches ofmicrocapsules with LbL membranes (solid lines). n is thestarting number of microcapsules in each batch.



turbed the original cellular function of the encapsu-lated cells.

CONCLUSION

In summary, a thorough investigation on the LbLadsorption process for the preparation of a PSS/PAH polyelectrolytes multilayer membrane on theconventional AP microcapsules has resulted in thesuccessful formation of robust and stable microcap-sules around cell spheroids prepared from animmortal cell line. It has been clearly demonstratedthat the conventional AP microcapsules are incapa-ble of providing durable support and protection forcell spheroids prepared from an immortal cell line,where a destructive pressure is generated on theencapsulating membrane because of continuous in-ternal cell proliferation. This structural instability isa major limiting factor for the survival of the micro-capsule device in transplantation applications.

The proposed LbL PSS/PAH membrane has beensuccessfully implemented. The construction of theLbL membrane has been subjected to electrophore-sis, and the Cryo-SEM and TEM imaging data haveclearly shown that the LbL membrane has beenassembled and modified on the surface of the con-ventional AP microcapsules. However, more experi-ments are required to quantitatively characterize themechanical properties of the LbL membrane.

The membrane durability study using the HEK293 cell line showed that the implementation of theLbL membrane on the conventional AP microcap-sules facilitated a significant improvement in struc-tural stability and resistance to internal stress. Theapplicability of the proposed device for drug deliv-ery and tissue transplantation was also demon-strated with the glucose-induced insulin secretionresponse from the microencapsulated MIN-6 cellline. The LbL membrane was shown to have similarpermeability and transport properties to the conven-tional AP microcapsules. No damage and deteriora-tion of the microencapsulated cells was observedfrom the process of assembling the LbL membraneand eventual incorporation of the membrane.

The authors thank Dr. Kim Sewell and Mr. Rick Webb,Centre for Microscopy and Microanalysis, University ofQueensland, for the help in performing the Cryo-SEM andTEM analysis.

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