Encapsulation of Basic Fibroblast Growth Factor by PolyelectrolyteMultilayer Microcapsules and Its Controlled Release for EnhancingCell ProliferationZhen She,†,‡,§ Chunxia Wang,† Jun Li,*,†,‡,§ Gleb B. Sukhorukov,§,∥ and Maria N. Antipina*,§

†Department of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574,Singapore‡NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of Singapore, Centre for Life Sciences, 28Medical Drive, Singapore 117456, Singapore§Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link,Singapore 117602, Singapore∥School of Engineering and Materials Science Queen Mary, University of London, Mile End Road, London, E1 4NS, UnitedKingdom

ABSTRACT: Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable toenvironmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by acoprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate andpoly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured byenzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with ashell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics,and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlledrelease for enhancing L929 cell proliferation was analyzed.

1. INTRODUCTIONBasic fibroblast growth factor (FGF2) is a mitogenic cytokineprotein (17.2 kDa), which regulates many aspects of cellularactivity, such as cell migration and extracellular matrixmetabolism. FGF2 stimulates growth and proliferation offibroblasts and capillary endothelial cells and enhances tissueregeneration and angiogenesis in the process of woundhealing.1−4 Moreover, FGF2 has been shown to maintainundifferentiated proliferation of human embryonic stem cells.5

FGF2 exhibits its optimal proliferative efficacy within acertain concentration range. For example, maximal rate ofproliferation of periodontal ligament cells has been observed atFGF2 concentration ranging from 10 to 100 ng/mL.6 However,FGF2 is highly vulnerable to environmental factors, especiallytemperature and pH. It degrades rapidly if surroundingtemperature is above 40 °C or pH is less than 5.7 When

injected in soluble form into a human body, efficacy of FGF2 islimited because of short retention time at wound sites and shorthalf-life caused by diffusion and susceptibility to enzymaticdegradation.8,9 Therefore, a suitable delivery system that canprotect and release FGF2 in a sustained and controlled manneris demanded.Currently available FGF2 delivery systems often suffer from

poor control of the capsule size and protein release rate, highdenaturation of the protein during the capsule fabrication, andlow loading efficiency. A number of hydrogel-based materials,for instance, chitosan−gelatin and alginate have been employedfor encapsulation and controlled release of FGF2.10,11

Received: April 16, 2012Revised: May 29, 2012Published: June 1, 2012

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Polyelectrolyte multilayer microcapsules have been versatileand multifunctional carriers for macromolecules.12,13 A varietyof loading routines have been developed for effectiveencapsulation of different active compounds.14 Layer-by-layer(LbL) encapsulation technology is also one of the most suitableand simplest methods to prepare microcapsules with tunablepermeability by varying their composition and thickness.15

pH is a well-known trigger to increase permeability ofpolyelectrolyte multilayer capsules.16−19 Itoh et al. preparedmicrocapsules via LbL assembly of chitosan (CT) and dextransulfate (Dex) and demonstrated postloading of FGF2 at pH 8,where the CT/Dex multilayers had a relatively highpermeability for the protein.20 Recently, postloading oftransforming growth factor-β1 (TGF-β1) into heparin/poly-L-arginine microcapsules with preloaded heparin was reported.These microcapsules could successfully maintain the bioactivityof TGF-β1 and stimulate myofibroblast differentiation andcontraction in vitro.21 We previously employed a proteinpreloading routine based on porous inorganic micrometer-sizedspherical templates presaturated with the protein molecules,followed by multilayer shell assembly and core extraction.22−24

This method has been proven to result in a higher loadingefficiency and slower release of bovine serum albumin (BSA)compared with postloading routine.15

Herein, we propose to use the preloading routine for FGF2encapsulation to achieve its sustained and controlled release forstimulating cell growth and proliferation. Another key issue inencapsulating and delivering FGF2 is to protect and maintainits biological activity. Therefore, we have coencapsulated FGF2with heparin, which is known to bind FGF2 and can maintainabout 80% bioactivity of FGF2 for 2 days at 37 °C. Under thesame conditions, FGF2 can only maintain less than 10%bioactivity if there is no heparin protection.2 In this report, wehave investigated the preloading routine for efficientencapsulating FGF2 and the coencapsulating heparin forkeeping the bioactive of FGF2. We measured the cytotoxicityof the microcapsules and obtained the FGF2 release profilesfrom the microcapsules. Finally, we performed a comparativestudy of the LbL microcapsule FGF2 delivery system and freeFGF2 in terms of the effect on proliferation of L929 cells.

2. EXPERIMENTAL SECTION2.1. Materials. All reagents were used as received without further

purification. Dextran sulfate sodium salt (Dex, MW > 500 000), poly-L-arginine hydrochloride (PAr, MW > 70 000), BSA, heparin (sodiumsalt from porcine intestinal mucosa), flourescein isothiocyanate bovineserum albumin (FITC-BSA), Dulbecco’s phosphate-buffered saline(DPBS), fetal bovine serum (FBS), penicillin, streptomycin, calciumchloride dihydrate, anhydrous sodium carbonate, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), and ethylenediaminetetraaceticacid trisodium salt (EDTA) were obtained from Sigma-Aldrich. Therecombinant human FGF2 (purity >95%) and Quantikine humanFGF basic immunoassay kit were purchased from R&D systems (cat.no.: DFB 50, ELISA). CellTiter 96 Aqueous nonradioactive cellproliferation assay (MTS) was purchased from Promega. Dulbecco’smodified Eagle’s medium (DMEM) was obtained from Invitrogen.Deionized water with specific resistivity higher than 18.2 MΩ·cm froma three-stage Milli-Q Plus 185 purification system was used in theexperiments.2.2. Loading of FGF2 into Polyelectrolyte Multilayer

Microcapsules. To obtain a stock solution, lyophilized powder ofFGF2 was reconstituted with DPBS buffer containing heparin and BSAto reach the final concentration of 100 μg/mL for FGF2 and a weightratio of BSA/heparin/FGF2 at 10:10:1. DPBS was used for furtherdilution. The FGF2 stock solution (1.0 mL) was mixed under vigorous

agitation with 1.0 mL of DI water, 1.0 mL of 1.0 M CaCl2 solution,and 1.0 mL of 1.0 M Na2CO3 solution. (All solutions were sterilized byfiltration with 0.2 μm filter.) The process resulted in the formation ofprotein-loaded CaCO3/FGF2 microparticles. The polyelectrolytemultilayer shell was formed via successive immersing of the CaCO3/FGF2 microparticles into solutions containing 0.5 M of NaCl and 2.0mg/mL of oppositely charged polyelectrolytes. Adsorption of eachpolyelectrolyte layer was followed by two consecutive washings withwater. Negatively charged Dex and positively charged PAr wereselected as shell constituents because of their biodegradability andgood mechanical stability of formed microcapsules due to tightcomplexation of the biopolymers.25,26 After the desired number ofpolyelectrolyte layers in the shell was achieved, CaCO3 template wasdissolved by three consecutive treatments with 0.2 M EDTA (pH 7.0)lasting for 30 min each. The obtained protein-loaded microcapsuleswere then washed two times with serum-free DMEM. By this routine,microcapsules comprising 6, 10, and 14 polyelectrolyte layers wereprepared and stored in a form of aqueous suspension at 4 °C ifnecessary. Supernatants obtained in each stage of the encapsulationprocess were collected for further analysis of the FGF2 concentration.The encapsulation efficiency of FGF2 was calculated as (mI − (mS1 +mS2 + ... + mSn))/mI × 100%, where mI is the initial amount of FGF2used in the process of CaCO3 synthesis and mS is the amount of FGF2in a supernatant.

The same protocol was followed for FITC-BSA loading intopolyelectrolyte multilayer capsules. Initial amount of FITC-BSA in 1.0mL of an aqueous solution used for CaCO3/FITC-BSA particlesynthesis was 1.0 mg.

2.3. Capsule Counting. The amount of capsules was determinedby a hemacytometer. Two units, “capsules per milliliter” (c/mL) and“capsules per cell” (c/c), were used in the manuscript to refer to thecapsule concentration in a sample. The amount of capsules in 1.0 mLof liquid comes directly from counting, and the conversion relationshipbetween the units is 2.5 × 106 c/mL = 100 c/c considering theprotocol of cell culture used in our experiments (described below).The routine of capsule synthesis described above usually resulted in∼9.0 × 108 capsules in the whole batch.

2.4. FGF2 Concentration Measurements. To minimize the lossof FGF2 caused by its adsorption onto the walls of test tubes and cellculture plates, we passivated the surfaces by BSA soaking overnight in1.0 mg/mL protein solution.

The Quantikine human FGF basic immunoassay kit (96 wells) wasused to measure the FGF2 concentration in the samples. TheQuantikine kit employs the enzyme-linked immunosorbent assay(ELISA), which is a widely accepted method for quantitative detectionof the bioactive FGF2.2 In brief, standards and samples were pipettedinto the microplate wells precoated with a monoclonal antibodyspecific for FGF2, and any FGF2 present was bound by theimmobilized antibody. After washing away any unbound substances,an enzyme-linked monoclonal antibody specific for FGF2 was addedto the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells, and colordeveloped in proportion to the amount of FGF2 bound in the initialstep. Absorbance was read at 450 nm by a microplate reader (TECANSpectrafluor Plus) with background subtraction at 570 nm. All datarepresented the mean of three measurements of three different trials,and results were reported as the means and standard deviations ofthese measurements.

2.5. FGF2 Release Profile Measurements. Measurements ofFGF2 release profiles were carried out in 96-well plates. DMEMmedium (200 μL) containing 5% FBS was added to each test well onthe first day. On the second day, the medium was refreshed by serum-free DMEM, and FGF2-loaded microcapsules were added. Super-natants were collected daily starting from the day when themicrocapsules were added (day 0, blank medium) to the third dayof incubation (day 3). The FGF2 concentrations in supernatants weremeasured by the Quantikine kit. All measurements were performed intriplicate.

2.6. Cell Culture Studies. L929 mouse fibroblast cell line(American Type Culture Collection, ATCC, Rockville, MD) was

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used to study the effect of the polyelectrolyte multilayer encapsulationon FGF2 delivery. The cells were incubated in 75 cm2 tissue cultureflasks at 37 °C in a humidified atmosphere of 5% CO2 and 95% airincubator before seeding in cell culture plates for further in vitroexperiments. The cell culture medium consisted of DMEM with 10%FBS, penicillin (50 U/mL), and streptomycin (0.05 mg/mL).To ensure cell proliferation is not affected by the space and

nutrition limitations, we performed a set of screening tests todetermine the optimal conditions for cell culture: the cell density was5000 cells per well in a 96-well plate; the volume of the medium was200 μL; and experiment duration without medium refreshment was 3days. To attenuate the denaturing rate of free FGF2 at 37 °C, weadded heparin to the cell culture medium in the weight ratio ofheparin: FGF2 = 10:1.For the purpose of attachment, L929 cells were seeded in wells filled

with 5% FBS DMEM. One day after (day 0), the medium was replacedwith serum-free DMEM, and tested dispersions of microcapsules orFGF2 solutions were added. The effect of FGF2 on cell proliferationwas evaluated three days later (day 3) using the calorimetric MTSassay designed to measure the activity of a dehydrogenase enzyme inmetabolically active cells. The absorbance corresponding to theamount of active cells was measured at 492 nm by a microplate reader.2.7. Optical Microscopy. FGF2-induced changes in cell

morphology were observed by Olympus IX51 microscope equippedwith 20× objective lens in a bright-field mode. Images were capturedat three different sites of each sample, and the ratio of the cells withapparent morphology changes to the cells with intact morphology wascalculated.2.8. Confocal Laser Scanning Microscopy (CLSM). FITC-BSA-

loaded microcapsules were incubated with L929 cells in a glass basedish. The cells were washed twice with DPBS buffer after 1 day ofincubation to remove free microcapsules that were not internalized bycells. Then, the cells were fixed in 4% paraformaldehyde for 15 min,and the cell nucleus was stained with DAPI for 5 min before CLSMmeasurements. Images were obtained on a Carl Zeiss Lsm510 METACLSM system (Carl Zeiss AG, Germany) equipped with a C-Apochromat 63×/1.2 Water Lens objective. The excitation wavelengthλex = 488 nm and emission wavelength λem = 525 nm were used forscanning of FITC-BSA, whereas λex = 405 nm and λem. = 430 nm wereused for DAPI. The depth scanning mode along the Z axis had aresolution of 0.7 μm/layer, and the pinhole size was 140 μm.

3. RESULTS AND DISCUSSION

3.1. FGF2 Encapsulation. Being sensitive to the environ-mental conditions, FGF2 loses its bioactivity if there is noproper protection.2 In our experiments, FGF2 lost thebioactivity almost completely during the CaCO3-assistedencapsulation process, probably due to prolonged exposure toroom temperature (more than 3 h), interaction with Ca2+

cations and components of cell culture medium, and substantialpH change caused by Na2CO3. To minimize FGF2denaturation, we used a buffer solution containing heparinand BSA as the aqueous medium for synthesis of FGF2/CaCO3microparticles. To verify the bioactivity of the incorporatedFGF2, we treated FGF2/CaCO3 microparticles with 0.2 M

EDTA (pH 7.4), and then the FGF2 concentration in thesolution after CaCO3 decomposition was examined with theQuantikine kit. Our measurements revealed that the loss ofFGF2 bioactivity upon CaCO3 microparticle synthesis did notexceed 5%.Table 1 summarizes the main parameters during the loading

and encapsulation of FGF2 into the Dex/PAr microcapsules ofdifferent thicknesses. The efficiency of the FGF2 loading intothe CaCO3 microparticles was 68%. The amount of recoveredFGF2 in the supernatants collected after adsorption of eachshell layer and upon particle washings indicates a moderate lossof preloaded FGF2 in the process of multilayer shell assembly,and it was in inverse relation to the number of layers adsorbed.For instance, deposition of the first polymer layer resulted in aloss of 9.6 μg of FGF2, whereas only 0.1 μg was lost duringdeposition of the fifth layer. The loss of FGF2 becamenegligible after the deposition of the sixth shell layer (Figure 1).Our results showed that 53% of the initial amount of FGF2 wasencapsulated in the FGF2/CaCO3 core−shell microparticles(Table 1).

Extraction of the CaCO3 template by EDTA caused burstrelease of a major fraction of the incorporated protein. The rateof the burst release could be controlled by the thickness of thepolyelectrolyte shell.15 In this study, capsules coated with thethicker shells lost less amount of FGF2 in the process ofCaCO3 dissolution (Table 1). For instance, the capsules withthe shell comprising 14 polyelectrolyte layers retained 42%FGF2, which was 13% more than those comprising 6 layers.The FGF2 encapsulation efficiency of this method (42%) ishigher than that based on CT nanoparticles (27%) previouslyreported.27 In another report, the efficiency of TGF-β1postloading into heparin containing polyelectrolyte multilayermicrocapsules was 35%.21

3.2. Optimal Concentration Range Of FGF2 For L929Cell Proliferation. To determine the optimal concentration

Table 1. Loading and Encapsulation of FGF2 into Dex/PAr Microcapsules of Different Thicknesses

shell thickness, numberof layers

initial amount ofFGF2 (μg)a

FGF2 in CaCO3microparticles (μg)b

FGF2 in core−shellmicroparticles (μg)

FGF2 inmicrocapsules (μg)c

encapsulationefficiency (%)d

6 100 68.0 ± 2.4 53.9 ± 2.0e 29.4 ± 5.0 29.4 ± 5.010 100 68.0 ± 2.4 f 35.9 ± 4.2 36.9 ± 4.214 100 68.0 ± 2.4 f 42.4 ± 4.6 42.4 ± 4.6

aInitial amount of FGF2 was calculated based on dilution rate of the stock solution. bMeasured by Quantikine kit from three samples. Capsules ofdifferent layers used the same batches of CaCO3 cores.

cMeasured by Quantikine kit from three samples. dRatio between FGF2 in microcapsules andinitial amount of FGF2. eMeasured by Quantikine kit for six-layer Dex/Par capsules from three samples. fFrom layer 6, the loss of FGF2 wasnegligible (Figure 1), so FGF2 in the 10- and 14-layer core−shell microparticles could be considered to be the same as that of 6-layer (54 μg).

Figure 1. Cumulative loss of FGF2 during the multilayer shellassembly.

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range of FGF2, we cultured L929 cells in the medium withdifferent concentrations of free FGF2. The results of the MTSassay performed after 3 days of cell culture are shown in Figure2. The rate of cell proliferation reached maximum at the FGF2

concentrations ranging from 10 to 100 ng/mL, where the cellnumber increased by ∼50% with respect to the control groupof cells cultured without FGF2 (Figure 2). A similar optimalconcentration range of FGF2 was reported for humanperiodontal ligament fibroblasts.6

3.3. Cytotoxicity of Dex/PAr Microcapsules. Forcytotoxicity studies, L929 cells were cultured for 3 days inthe presence of hollow Dex/PAr microcapsules of differentthicknesses. The results shown in Figure 3 suggest that the

microcapsules had low cytotoxicity at concentrations lowerthan 50 c/c. Above this concentration, cell viability droppedbelow 80%, indicating a considerable negative effect of thecapsules on cell proliferation. Importantly, the shell thicknesshad a negligible impact on the capsule cytotoxicity.3.4. In Vitro Release Kinetics of FGF2 from Dex/Par

Microcapsules. We reported that the kinetics of protein(BSA) release from the polyelectrolyte multilayer micro-capsules depended on the capsule thickness and concen-tration.15 Therefore, release profiles that match the optimalFGF2 concentration range could be achieved by varying of thecapsule thickness and concentration. Herein, we first measuredthe release profiles for capsules composed of three, five, andseven Dex/PAr bilayers at 7.5 × 105 c/mL (equivalent to 30 c/c in our cell culture protocol), which showed negligiblecytotoxicity (Figure 4a). The FGF2 release was performed in

serum-free DMEM. The supernatants were collected daily over3 days; then, the FGF2 concentrations were measured by theQuantikine kit. The FGF2 concentration reached maximumlevels for (Dex/PAr)3 microcapsules on day 1 and for (Dex/PAr)5 and (Dex/PAr)7 microcapsules on day 2, after which theFGF2 concentration decreased. On day 3, Dex/PAr micro-capsules with thicker shells maintained higher FGF2 concen-trations than those with thinner shells. FGF2 was protected byheparin in our experiments.The main concern about the release profiles in Figure 4a was

the very low detected bioactive FGF2 concentrations in themedium. In an attempt to match the optimal FGF2concentrations, we increased the amount of the capsules to100 c/c (Figure 4b). The release profiles in Figure 4b followedthe same trend as those in Figure 4a, and only (Dex/PAr)7capsules at the concentration of 100 c/c could achieve theoptimal FGF2 concentrations for L929 cell proliferation.

3.5. Controlled Release of FGF2 from Dex/ParMicrocapsules for Enhancing L929 Cell Proliferation.To figure out whether the controlled release of FGF2 bypolyelectrolyte microcapsules has advantages over the freeFGF2 on L929 cell proliferation, L929 cells were cultured inthe presence of FGF2-loaded (Dex/PAr)3, (Dex/PAr)5, and(Dex/PAr)7 microcapsules with concentrations falling into therange of acceptable cytotoxocity (from 1 c/c to 100 c/c). Cellnumbers after 3 days of incubation with FGF2-loadedmicrocapsules were measured with respect to a control withL929 cells cultured in the medium containing 10 ng/mL of freeFGF2, which is the optimal condition for L929 cell proliferationwithout a controlled release system. Despite the release profiles,the results showed that the microcapsules were capable topromote L929 cell proliferation at capsule concentrationsranging from 5 to 30 c/c, providing 10−30% increase in cellnumber after 3 days of culture (Figure 5a). At the same time,we did not observe any significant difference between themicrocapsules of different thicknesses.We also plot the relative proliferation of L929 cells after 3

days culture with free FGF2 (10 ng/mL), hollow (Dex/PAr)3

Figure 2. Effect of free FGF2 on proliferation of L929 cells.

Figure 3. Cell number after three days of culturing in serum-freeDMEM in the presence of hollow Dex/PAr capsules comprising threebilayers (-□-), five bilayers (-○-), and seven bilayers (-△-).

Figure 4. In vitro release of FGF2 from (Dex/PAr)3 (-□-), (Dex/PAr)5 (-○-), and (Dex/PAr)7 (-△-) microcapsules into 200 μL ofserum-free DMEM at 37 °C. (a) Capsule number: 1.5 × 105.Concentration of capsules: 7.5 × 105 c/mL (or 30 c/c), equivalent tototal FGF2 concentrations of 24, 30, and 35 ng/mL for (Dex/PAr)3,(Dex/PAr)5, and (Dex/PAr)7, respectively. (b) Capsule number: 5 ×105. Concentration of capsules: 2.5 × 106 c/mL (or 100 c/c),equivalent to total FGF2 concentrations of 80, 100, and 117 ng/mLfor (Dex/PAr)3, (Dex/PAr)5, and (Dex/PAr)7, respectively.

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capsules (10 c/c), and FGF2-loaded (Dex/PAr)3 capsules (10c/c) in Figure 5b. Here the relative cell numbers are calculatedwith respect to the control group with cells cultured in DMEMwithout the addition of any FGF2 or capsules. Whereas the freeFGF2 resulted in 150% cell proliferation, FGF2-loaded (Dex/PAr)3 capsules could further increase the cell proliferation tonearly 200%. The group with added hollow capsules showednearly the same cell proliferation rate as the control group,indicating that the cytotoxicity of the hollow capsules isnegligible.Another evidence of effective controlled release of FGF2 by

the polyelectrolyte multilayer microcapsules was obtained byinvestigating the cell morphology (Figure 6). Although FGF2 isthought to be the most important protein to promote mitoticactivity and cell proliferation, it is also known to suppresssynthesis of collagen I and III, causing an abnormal cellmorphology.28−30 We found that over 80% of L929 cells in abatch turned round after 2 days of culture in the mediumcontaining 10 ng/mL of free FGF2. Similar result was obtainedfor a group of cells incubated with 5−30 c/c FGF2-loadedDex/PAr microcapsules, whereas <5% of cells exhibited theabnormal spherical morphology in a control group cultured inserum-free DMEM or in the presence of hollow Dex/PArmicrocapsules.Although the released FGF2 concentration levels in the

supernatants were lower than those optimal FGF2 concen-trations for L929 cell proliferation in free FGF2 systems, theuse of FGF2-loaded microcapsules resulted in 10−30% highercell proliferation rate after 3 days of culture (Figure 5). Thepossible reason may be the protection of FGF2 that was storedin the interior of the polyelectrolyte multilayer shells, and each

portion of released FGF2 was absorbed by the cells before itsdegradation. To verify this assumption, we measured theconcentrations of free and encapsulated FGF2 in the mediumwith and without cells and then calculated the amounts ofbioactive FGF2 in the total volume of the medium in a well(200 μL) (Figure 7). The difference of the areas under the

curve measured without cells and that with cells can beconsidered to be the amount of FGF2 absorbed by the cells.The total absorbed FGF2 amount in the presence of (Dex/PAr)7 capsule system was five times higher than that in the freeFGF2 system. Therefore, the use of polyelectrolyte micro-capsules for FGF2 release could reduce the overall amount ofFGF2 needed in the cell culture, providing the same or evenhigher rate of cell proliferation. For the optimal cellproliferation, free FGF2 system required higher FGF2 dosages(2−20 ng), whereas the microcapsule-controlled releasesystems required much less FGF2 dosages (0.8−4.8 ng for(Dex/PAr)3, 1.0−6.0 ng for (Dex/PAr)5, and 1.2−7.0 ng for(Dex/PAr)7).Another possible reason for the enhancement of cell

proliferation by the encapsulated FGF2-controlled releasesystem could be high affinity of the polyelectrolyte micro-capsules to the cell surface. Figure 8 displays CLSM images of

Figure 5. (a) Relative numbers of L929 cells after 3 days of culturewith FGF2-loaded capsules of different concentrations and shellthicknesses (Dex/PAr)3 (-□-), (Dex/PAr)5 (-○-), and (Dex/PAr)7(-△-) with respect to the control with 10 ng/mL of free FGF2 (asrelative cell number 100%); **: P < 0.05 (from two-tail student t test)indicates significant difference from control group. (b) Relativenumbers of L929 cells after 3 days of culture with free FGF2 (10 ng/mL), hollow (Dex/PAr)3 capsules (10 c/c), and FGF2-loaded (Dex/PAr)3 capsules (10 c/c) with respect to the control group (as relativecell number 100%) with cells cultured in DMEM without the additionof FGF2 or capsules.

Figure 6. Cell morphologies after two days of culture in the presenceof 10 ng/mL of free FGF2 (ca. 84% of spherical cells) (a), in serum-free DMEM (ca. 2% of spherical cells) (b), in the presence of FGF2-loaded (Dex/PAr)3 capsules (5 c/c) (ca. 82% of spherical cells) (c),and in the presence of hollow (Dex/PAr)3 capsules (ca. 1% ofspherical cells) (d). The percentage of cells with spherical morphologyis the average from three different wells.

Figure 7. Amounts of bioactive FGF2 detected in DMEM mediumupon incubation at 37 °C: free FGF2 without cells (-○-) and with cells(-□-) and (Dex/PAr)7-encapsulated FGF2 without cells (-●-) andwith cells (-■-) (capsules concentration: 30 c/c).

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cells in the presence of Dex/PAr microcapsules loaded withfluorescence-labeled BSA (FITC-BSA). It can be seen that themicrocapsules could attach onto the cell surface (Figure 8a,b).Moreover, from the 3D images (Figure 8c), some micro-capsules were found to be internalized by cells. This feature ofthe polyelectrolyte microcapsules may become especiallyadvantageous in vivo because free FGF2 has very shortretention time at the wound site due to fast diffusion andenzymatic degradation.31 Because of high affinity to the cellsurface, the microcapsules are expected to create higher localconcentration of FGF2 at the wound site, enabling moreeffective utilization of FGF2 by the cells.Although FGF2 is essential to promote cell growth, we

observed that the number of cells cultured in 10% FBS with noextra FGF2 added can reach almost 200% proliferation rate ascompared with the control group of cells cultured in themedium containing 10 ng/mL of free FGF2. It was found thatthe 10% FBS medium contained only ∼8 pg/mL of FGF2,indicating that other actives and compounds in the medium arealso important for the cell proliferation. Indeed, in addition toFGF2, a number of growth factors regulating cell division andcell survival can be found in human serum, including insulin-like growth factor, platelet-derived growth factor, epidermalgrowth factor, and TGF-β. The CaCO3 presaturation routinemay enable simultaneous loading of different growth factorsinto the polyelectrolyte multilayer microcapsules, and therelease multiple growth factors from the microcapsules may bepromising for would-healing therapies. For example, it wasreported that normal extracellular matrix formation is essentialfor scar-free wound healing. Release of platelet-derived growthfactor and TGF-β can promote the normal extracellular matrixformation.28−30 Future studies may also involve protection ofFGF2 against the oxidative stress inside cells by furthermodification of microcapsules.32

4. CONCLUSIONSPolyelectrolyte multilayer microcapsules were employed forcontrolled release of basic FGF2 in vitro. Because FGF2 ishighly vulnerable to the environmental conditions, such astemperature, pH, salinity, and enzymes, special attention wasgiven to maintain its bioactive form upon encapsulation andrelease. Encapsulation of FGF2 into dextran sulfate/poly-L-arginine (Dex/PAr) shells of different thicknesses wasperformed by preloading of the protein into porous CaCO3microparticles, followed by shell assembly and extraction of theinorganic template. Heparin and BSA were simultaneouslyloaded into the microcapsules and proved to protect the

bioactivity of FGF2 in each stage of the encapsulation processand upon storage inside the capsules.The Dex/PAr microcapsules were low cytotoxic for L929 cell

line and capable to sustain release of the incorporated protein.The FGF2 release rate was finely adjusted by changing thethickness of the microcapsules.The controlled release of FGF2 from the microcapsules was

advantageous to enhance the proliferation of L929 cells overthe system with free FGF2. In particular, the controlled releasesystems required much lower FGF2 concentration for achievingeven higher cell proliferation rate than the free FGF2 systems.Presumably, good protection of FGF2 in the interior of thepolyelectrolyte multilayer shells and high affinity of themicrocapsules to the cell surface facilitated utilization of theprotein by cells minimizing the undesired denaturation ofFGF2 in the solution.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +65 6516 7273. Fax: +65 6872 3069. E-mail: bielj@nus.edu.sg (J.L.); antipinam@imre.a-star.edu.sg (M.N.A.).Author ContributionsZ.S. and C.W. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was financially supported by Science and Engineer-ing Research Council, Agency for Science, Technology andResearch (A*STAR), Singapore (grant no. PSF 102 101 0024),and Institute of Materials Research and Engineering, A*STAR(IMRE/08-1C0204 and IMRE/09-1C0321). Z.S. thanks NUSGraduate School for Integrative Sciences and Engineering(NGS) of National University of Singapore for the NGSGraduate Scholarship.

■ REFERENCES(1) Langer, R.; Vacanti, J. P. Science 1993, 260, 920−926.(2) Caldwell, M. A.; Garcion, E.; terBorg, M. G.; He, X.; Svendsen, C.N. Exp. Neurol. 2004, 188, 408−420.(3) Sommer, A.; Rifkin, D. B. J. Cell. Physiol. 1989, 138, 215−220.(4) Tsuboi, R.; Rifkin, D. B. J. Exp. Med. 1990, 172, 245−251.(5) Xu, R. H.; Peck, R. M.; Li, D. S.; Feng, X.; Ludwig, T.; Thomson,J. A. Nat. Methods 2005, 2, 185−190.(6) Takayama, S.; Murakami, S.; Miki, Y.; Ikezawa, K.; Tasaka, S.;Terashima, A.; Asano, T.; Okada, H. J. Periodontal Res. 1997, 32, 667−675.

Figure 8. CLSM images of L929 cells incubated with FITC-BSA-loaded (Dex/PAr)3 microcapsules: (a) Cross-section fluorescence image with z-axisfluorescence projection at the cross-plane displayed (windows at the bottom and on the right), scale-bar: 20 μm. (b) Overlap of fluorescence modeand bright-field mode, scale-bar: 20 μm. (c) 3D fluorescence image from the top view, frame length, and width: 210 μm; height: 13 μm.

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