European Polymer Journal 44 (2008) 1654–1661

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Contents lists available at ScienceDirect

European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Macromolecular Nanotechnology

Preparation, characterization and biocompatibility ofpoly(ethylene glycol)-poly(n-butyl cyanoacrylate) nanocapsuleswith oil core via miniemulsion polymerization

Yu Zhang a,b, Siyu Zhu a,b, Lichen Yin a,b, Feng Qian a,b, Cui Tang a, Chunhua Yin a,b,*

a State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, Chinab Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 October 2007Received in revised form 14 March 2008Accepted 17 March 2008Available online 30 March 2008

Keywords:Poly(butylcyanoacrylate)Poly(ethylene glycol)PaclitaxelNanocapsules

0014-3057/$ - see front matter � 2008 Elsevier Ltddoi:10.1016/j.eurpolymj.2008.03.019

* Corresponding author. Address: State Key LaEngineering, Department of Pharmaceutical ScienSciences, Fudan University, Shanghai 200433, Chin3797; fax: +86 21 5552 2771.

E-mail address: chyin@fudan.edu.cn (C. Yin).

A new type of nanocapsules with an oil core, coated by poly(ethylene glycol) (PEG) wasdesigned. The loading efficiency and the biocompatibility of the polymeric nanocapsuleswere evaluated when it was used as a carrier for hydrophobic agent paclitaxel. Thenanocapsules were synthesized through miniemulsion polymerization of butylcyanoac-rylate (BCA) with PEG as initiator. The particle size and zeta potential of nanocapsuleswere influenced by the PEG content in the polymerization system. Fourier transforminfrared (FTIR) spectra and 1H NMR demonstrated the chemical coupling between PEGand poly(butylcyanoacrylate) (PBCA). Thermal characteristics of the copolymer wereinvestigated by differential scanning calorimetry (DSC). The encapsulation efficiencyincreased concurrently with the increase of the PEG content in the system. The hemo-lytic assay and the cytotoxicity measurement showed that the PEG coating could signif-icantly reduce the hemolytic potential and cytotoxicity of the nanocapsules. The resultsshowed that the PEG–PBCA nanocapsules could be an effective carrier for hydrophobicagents.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Alkylcyanoacrylates (ACAs) have been proven to bevaluable monomers for several biomedical applicationsdue to their excellent adhesive properties, high reactivity,and good tissue tolerability. N-butylcyanoacrylate (Inder-mil�, liquiband�) is used clinically in Europe, Canada andthe USA, and the octylcyanoacrylate (Dermabond�) re-ceived Food and Drug Administration approval in 1998and now has been marketed in the USA [1]. They were usedextensively as tissue adhesives for the closure of skin

. All rights reserved.

boratory of Geneticces, School of Life

a. Tel.: +86 21 6564

wounds, surgical glue, and embolitic material for endova-sculatar surgery, which were based on the fact that the an-ionic polymerization could be easily initiated by traces ofnucleophiles like water, amines, alcohols, or phosphines.During the last decade, the polymeric nanoparticles pre-pared by poly(alkylcyanoacrylates) (PACAs) have gainedincreasing interest due to their biocompatibility and biode-gradability, and were reported to show a distinct tendencyfor the encapsulation of various pharmacologically activeagents, such as cytotoxic drugs [2], antibiotics [3], peptide[4,5] and gene [6]. These nanoparticles, including nano-spheres and nanocapsules, are satisfactory drug deliverycarriers. And some of them such as polyisohexylcyanoacry-late nanoparticles carried doxorubicin have already en-tered phase I clinical trial for cancer therapy [7].

The common methods prepared alkylcyanoacrylatenanoparticles involve emulsion polymerization, interfacial

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polymerization and interfacial precipitation. In the con-ventional emulsion polymerization, drugs are dissolved inthe polymerization system before introducing monomersor added after the polymerization so that drugs are encap-sulated during polymerization or adsorbed in the particles[8]. However, some highly hydrophobic agents like paclit-axel, the solubility of which in water is less than 3 lg/ml,are impracticable to be loaded efficiently. A convenientway to solve the problem is the application of the mini-emulsion technique in which the polymerization is initi-ated after a stable miniemulsion has been formed inmedium containing stabilizers. Miniemulsion is a relativelystable dispersion of oil droplets in water in the form of par-ticles with sizes between 30 and 500 nm, prepared byshearing a system of oil, water and surfactant. The resultantoil-core nanocapsules could improve the drug loading effi-ciency significantly [9]. Recently, the miniemulsion processhas been adapted to the anionic polymerization of BCA, andfunctionalization of the particle surface and the adjustmentof the particle size could be accomplished by using theminiemulsion technique [10].

As one of the most commonly investigated hydrophilicpolymers, poly (ethylene glycol) (PEG) is able to modifythe surface of particulate carriers. The PEG-coated nano-particles presented fine potential in therapeutic applica-tion for controlled release of drugs and site-specificdrug delivery. Some studies showed that nanoparticlesprepared by emulsion polymerization of alkylcyanoacry-lates in the presence of PEG represented potential stealthcarriers, possibly able to avoid the recognition of themononuclear phagocyte system (MPS) in vivo [11–13].According to the previous studies, the –OH groups (hy-droxyl groups) of PEG were deemed as initiators ofalkylcyanoacrylate polymerization, thereby forming thecovalent bond between PEG and PACA [14], and the cel-lular uptake kinetics and targeting abilities of nanoparti-cles could be influenced by the chemical modification[15].

In this study, PEG modification and miniemulsion tech-nique were combined to provide an efficient carrier forhydrophobic drugs. Medium-chain triglyceride was usedto form the oil cores in aqueous medium and n-butylcy-anoacrylate monomers were distributed around them.Methoxypoly(ethylene glycol) (mPEG) acted as a nucleo-phile initiator of the polymerization of n-butylcyanoacry-late through its –OH (hydroxyl) terminal group, thusforming PEG–PBCA chains that surrounded on the surfaceof oil cores by hydrophobic interaction. As a result, a newtype of nanocapsule with oil core and PEG–PBCA outerlayer was obtained. Fourier transform infrared spectros-copy (FTIR) and 1H NMR spectroscopy were employed toexamine the chemical structure of the formed polymer.Thermal characteristics of the copolymer were also inves-tigated by differential scanning calorimetry (DSC). PEG–PBCA nanocapsules were characterized by analysis ofparticle size, zeta potential and morphology. The hydro-phobic drug paclitaxel was used as a model drug to beloaded and the encapsulation efficiencies were investi-gated. Moreover, the hemolysis and MTT assay were per-formed to estimate the biocompatibility of the polymericnanocapsules.

2. Materials and methods

2.1. Materials

Methoxypoly(ethylene glycol) (mPEG, MW5000 Da and2000 Da) was obtained from Sigma (USA). N-butylcyanoac-rylate (BCA) monomers were obtained from Beijing SunconMedical Adhesive Company. Acetonitrile (Merck, Ger-many) was of HPLC grade. All the other reagents were ana-lytical grade. Water used was double distilled.

2.2. Preparation of nanocapsules

The PEG–PBCA nanocapsules were prepared by theminiemulsion polymerization method. Firstly, 0.1 g ofmedium-chain triglyceride Miglyol 812 was added to10 ml of an aqueous solution of 0.1 mol/L hydrochloric acid(pH 1.0) containing different amounts of mPEG, and soni-cated for 75 s to yield a pre-emulsion. Then, 100 ll ofBCA monomers (about 0.7 mmol) was added into thepre-emulsion under mechanical stirring. The two-phasemixture was sonicated for 75 s to obtain a milky whiteemulsion and then stirred for 4 h. When the paclitaxel-loaded nanocapsules were prepared, paclitaxel was dis-solved in the BCA monomers then added into the pre-emulsion. And the next steps were the same as the blanknanocapsules.

To study the effect of the content of PEG on the physico-chemical properties of PEG–PBCA nanocapsules and theirdrug loading capability for paclitaxel, the concentration ofmPEG2000 or mPEG5000 in acid aqueous solution variedfrom 2% to 10% (w/v). The following nomenclature wasadopted for the polymers: (X) PEG–PBCA, where X denotesthe amount of PEG (namely 2%, 5% and 10% PEG–PBCA). Inaddition, aqueous solutions of 0.01 mol/L and 0.001 mol/Lhydrochloric acid (pH 2.0 and pH 3.0) were used to investi-gate the effect of the pH value of the polymerization systemon the synthesis of the polymeric nanocapsules.

In comparison with PEG–PBCA nanocapsules, paclit-axel-loaded PBCA nanoparticles were prepared as follows.Briefly, the BCA monomers were added into 0.1 mol/Lhydrochloric acid solution containing 0.5% pluronic F68as stabilizer, and the system was then sonificated andmagnetic stirred for the same time as the PEG–PBCA nano-capsules. For the PEG–PBCA nanocapsules and PBCA nano-particles, the concentration of paclitaxel fed in monomerwas 1% (w/w).

2.3. Chemical characterization and thermal analysis

To characterize the chemical structure of the PEG–PBCApolymer, the polymer was synthesized through the sameprocedure as the nanocapsules except adding the oil andsonicating to form a pre-emulsion. After dialysis, thePEG–PBCA polymer was collected through centrifugationat 12,000 rpm (about 10,000 g) for 10 min and washedfor several times, then lyophilized. The lyophilized poly-mer was characterized by FTIR and 1H NMR. FTIR spectrumwas obtained on a Thermo Nicolet Nexus 470 FTIR spec-trometer (USA). 1H NMR (JNM-MY60FT, JEOL, Japan) spec-

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trum was obtained using CDCl3 as solvent and tetrameth-ylsilane (TMS) as internal reference.

Thermal characteristics of mPEG, PBCA, the mixture ofmPEG and PBCA, and PEG–PBCA copolymer were investi-gated by DSC on a TA Instruments 2910 DSC (USA). Themeasurements of lyophilized polymer samples were per-formed from 25 �C to 220 �C at a heating rate of 10 �C/min.

2.4. Morphology of nanocapsules

The morphological examination of the nanocapsuleswas performed using transmission electron microscope(TEM) (H-600A, Hitachi, Japan) and scanning electronmicroscope (SEM) (SEM515, Philip, USA). For TEM observa-tion, the properly diluted nanocapsule suspension wasmounted on a copper grid and treated by negative stainingwith sodium phosphotungstate solution (0.2%, w/v). BeforeSEM observation, the nanocapsules were dried in vacuo at40 �C and coated with gold for enhancing the surfaceconductivity.

2.5. Analysis of particle size and particle surface charge

Dynamic light scattering measurements were carried outat 25 �C using a Zeta Potential/Particle Sizer (NicompTM380ZLS, USA). Scattered light with a wavelength of 635 nmwas detected at an angle of 90�. The nanocapsule suspensionwas diluted with hydrochloric acid appropriately for opti-mum measurement. The average hydrodynamic particlesize was expressed as the value of average size ±SD fromthree replicated samples. The width of the size distributionwas indicated by the polydispersity index (PI).

2.6. Drug encapsulation efficiency

The paclitaxel-loaded PEG–PBCA nanocapsules andPBCA nanoparticles were collected through centrifugationat 12,000 rpm for 20 min. The supernatant was removedand the particles accumulated at the bottom were dis-solved in acetonitrile and the solution was filtered through0.45 lm membrane, then 20 ll of the filtered solution wasinjected into a high performance liquid chromatographic(HPLC) apparatus. The HPLC apparatus was equipped witha Simadzu LC-10AD pump, a UV detector (Simadzu, SPD-10VP) operated at a wavelength of 227 nm, and a HypersilC18 column (5 lm, 150 mm � 4.6 mm, Yilite, China). Themobile phase was acetonitrile/water (50/50, v/v) andthe flow rate was 1.0 ml/min. Results were expressed asthe means of three measurements. The encapsulation effi-ciency (EE) was defined as follows

EE ð%;w=wÞ¼Mass of drug in nanoparticles=Mass of feed drug� 100

2.7. Biocompatibility studies of the nanocapsules

2.7.1. Hemolytic potential of nanocapsulesThe hemolytic potential of nanocapsules was investi-

gated according to the reported procedures [16]. In brief,

the fresh dog erythrocytes were separated through centri-fugation and washed several times with physiological sal-ine to remove residual blood plasma. Two milliliters ofpurified erythrocytes were resuspended with 38 ml of sal-ine to adjust a final hematocrit of 5%. Then 0.4 ml of theresulting erythrocyte suspension was incubated with0.8 ml of PBCA nanoparticle suspension or PEG–PBCAnanocapsule suspension of various concentrations in salineunder 37 �C and the system was shaken with a thermalconstant bath shaker (THZ-C, Jiangsu, China) at 300 rpmfor 1 h. Then the nanocapsules were separated throughthe centrifugation at 12,000 rpm for 20 min. The superna-tant of the samples exhibited light red while the superna-tant of the negative control appeared clearly transparentwhich indicated that the red blood cells were not damagedduring the centrifugation. The content of hemoglobin re-leased from disrupted erythrocytes was determined photo-metrically at 541 nm in the supernatant. Erythrocytesincubated in physiological saline served as negative con-trols and physiological saline containing 10 mg/ml Triton100 was used as positive controls. The percentage of hemo-lyzed cells was calculated as follows

Hemolysis ð%Þ¼ ðoptical density at 541 nm ðOD541Þ of sample� OD541 of negative controlÞ=ðOD541 of positive control� OD541 of negative controlÞ � 100

2.7.2. MTT assayQGY7703 cells were seeded onto 96-well-plates at a

density of 10,000 cells/well using Dulbecco’s modified Ear-le’s minimal essential medium (DMEM). The cells werekept at 37 �C and 5% (v/v) CO2 in a cell culture incubatorfor 24 h prior to use. The cells were washed using phos-phate buffered saline (PBS) pH 7.4 and incubated withnanocapsule suspension with a final concentration of1 mg/ml cell culture medium. Four hours later, the med-ium was removed and the cells were washed with PBS,and 200 ll of DMEM medium and 20 ll of MTT solution(5 mg/ml) were added. After incubation for another 3 h,the supernatant was removed, and the cells were lysedby the treatment with 200 ll of dimethyl sulfoxide(DMSO). The quantity of the resulting blue dye was ob-tained from a microplate reader (Model 450, Bio-Rad,USA) at a determined wavelength of 490 nm and a refer-ence wavelength of 570 nm. Cells treated with DMEMthroughout served as negative control and were deemedas 100% viability.

3. Results and discussion

3.1. Preparation of nanocapsules

The process of miniemulsion polymerization was exhib-ited in Fig. 1. After ultrasonication of medium-chain tri-glyceride and monomers in 0.1 mol/L hydrochloric acidsolution, they were dispersed into stable droplets of sub-micronic sizes [17]. In order to avoid evaporation of themonomers, heating of the dispersion during ultrasonica-tion was minimized by using pulsed ultrasound and

Fig. 1. Synthesis scheme of mPEG–PBCA nanocapsules prepared via miniemulsion polymerization.

Fig. 2. FTIR spectrum of mPEG5000–PBCA polymer.

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cooling the system. The BCA monomers located on the sur-face of the droplets would be initiated by the nucleophilcPEG in aqueous medium [14].

Initiated with the PEG molecules, the PBCA chains wereextending along with the proceeding of polymerization.The formed polymer mPEG–PBCA possessed the amphi-pathic properties. At the oil–water interface, the estergroups of PBCA would attach to the oil core of medium-chain triglyceride Miglyol 812, while the PEG chain, withits hydrophilic and flexible nature, would form hydrophiliccoating outside the nanocapsules. Because of the stericrepulsion effect of mPEG, no further stabilizer was neededin this nanocapsule suspension.

The paclitaxel molecule is chemically stable and inert inacid or neutral medium, so it would not react with the BCAmonomers in the acid medium with the nucleophilc PEGexisted. Some researches have used the method of dissolv-ing paclitaxel in BCA and it is proved to be practicable [9].In the preparation of the paclitaxel-loaded nanocapsules,the paclitaxel in BCA monomer presented transparentappearance and remained unchanged before being addedinto the reaction system.

3.2. FTIR spectrum and 1H NMR spectrum analysis

The FTIR spectrum of mPEG–PBCA (Fig. 2) showedabsorption bands related to the -CN stretching vibrationat 2249 cm�1 and the ester carbonyl at 1751 cm�1. TheC–O–C of the mPEG appeared at 1066 cm�1, which verifiedthe presence of mPEG in the polymer.

The 1H NMR spectrum of the mPEG–PBCA is consistentwith the structure indicated in the FTIR spectrum. In Fig. 3,the resonance at 3.64 ppm was attributed to the mPEGbackbone methylene, whereas the signal at 3.39 ppm wasattributed to the MeO terminal group of the mPEG chains.Both of the peaks indicated that mPEG had been conju-gated to the polymer. The broad peak at 2.90–2.20 ppmwas assigned to the methylene protons of poly-(cyanoac-rylate). The peak at 4.30 ppm corresponded to the methy-lene in the a-position to the ester groups. Signals at 1.72,1.26, and 0.88 ppm were assigned to the methylene andmethyl protons of the butyl chains. Based on the 1H NMRspectrum, the ratio of mPEG to BCA was calculated to beabout 1:40, which suggested that each mPEG chain couldinitiate polymerization of 40 monomers averagely.

3.3. Thermal analysis

Fig. 4 showed the DSC thermograms of mPEG5000,PBCA, mixture of mPEG and PBCA, and mPEG–PBCA copoly-mer. mPEG5000 exhibited an endothermal melting transi-tion at 62.8 �C and PBCA exhibited an endothermal peakat 46.8 �C. Both of the endothermal peaks appeared in theDSC curve of the mixture of mPEG and PBCA. But mPEG–PBCA did not reveal the presence of mPEG and PBCAendothermal melting peaks, and there was only a gradualendothermal transition presented when the temperaturewas higher than 150 �C. The phenomena might be due tothat the copolymerization disfavored the crystallization ofPEG and the copolymer existed mainly in amorphousphase. The difference between mPEG–PBCA and the mix-ture of mPEG and PBCA revealed that a new copolymerhad been synthesized and the covalent binding betweenmPEG and PBCA occurred. Similar results of DSC had beenreported in the research on other copolymer [14,18].

3.4. Morphological characterizations

Transmission electron micrographs and scanning elec-tron micrograph of the paclitaxel-loaded nanocapsulesprepared with 5% mPEG5000 were shown in Figs. 5 and6. It could be seen that the nanocapsules dispersed witha bright oil core, surrounded by a dark fluffy coat (Fig. 5aand b), presumably consisting of the PEG chains in a brush

Fig. 3. 1H NMR spectrum of mPEG5000–PBCA in CDCl3.

Fig. 4. DSC thermograms of mPEG5000 (A), PBCA (B), mixture of mPE-G5000 and PBCA (C) and mPEG–PBCA (D).

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configuration [19]. From the SEM image (Fig. 6), the nano-capsules were globular with dimensional uniformity andthe outer layer of the nanocapsules could be observed.The capsular structure was especially suitable for the loadof the hydrophobic compounds and the polymer shell aswell as the hydration layer could protect the incorporateddrugs from being released and help to achieve a probablesustained release.

3.5. Effect of mPEG on the particle size and zeta potentials

Table 1 showed the particle sizes, polydispersities, andzeta potentials of the mPEG–PBCA nanocapsules prepared

with mPEG2000 and mPEG5000 in hydrochloric acid. Theparticle sizes and zeta potential values were influencedby the quantity and the molecular weight of mPEG. WhenmPEG2000 was used, the particle sizes and the size distri-bution diminished as the concentration of mPEG2000 inthe medium increased which was in accordance with therole of the stabilizer of the system [20]. The mPEG mole-cules provided steric repulsion between the particles andhelped to obtain a stable dispersion. Regarding the nano-capsules prepared with mPEG5000, the particle sizes forall concentrations of mPEG were smaller and could befound within the range at around 200 nm. It could be con-cluded that, with the prolongation of the PEG chain length,mPEG5000 acted as a stronger stabilizer compared tomPEG2000. Additionally, with the increase of concentra-tion of mPEG in the suspension from 2% to 10% formPEG5000, improvement was achieved in the size distri-bution of nanocapsules.

Negative zeta potential was decreased when the con-centration of mPEG increased, which might be attributedto the nonionic properties of mPEG molecules overlayingthe surface of nanocapsules. The zeta potentials of nano-capsules prepared in hydrochloric acid solution of differentconcentrations using 2% mPEG5000 were also listed (Table1). The surface charges of nanocapsules prepared in0.1 mol/L hydrochloric acid solution significantly de-creased in comparison with those in 0.01 mol/L and0.001 mol/L hydrochloric acid solution, which demon-strated the presence of hydrophilic PEG chains on the sur-face of nanocapsules. Since alkylcyanoacrylates tend topolymerize extremely rapidly in the presence of moistureor traces of basic components, acid solution of high con-centration is necessary to limit the reaction speed andmake the polymerization process controllable. It was re-ported previously that the highest PEG association withPBCA was obtained at pH 1.0, where the ratio betweenmPEG –OH and water –OH was in favor of the formerone [14]. When the pH value was still lower, there would

Fig. 5. TEM of nanocapsules prepared with 5% mPEG5000 � 20,000 (a), and �50,000 (b). Bar represents 200 nm.

Fig. 6. SEM of nanocapsules prepared with 5% mPEG. Bar represents 500 nm.

Table 1Particle size and surface charge of paclitaxel-loaded PEG–PBCA nanopar-ticles prepared in 0.1 mol/L hydrochloric acid (n = 3)

PEG (% w/v) Size (nm) Polydispersity Zeta potential(mv)

2 (mPEG2000) 560.8 ± 6.6 0.066 �3.08 ± 0.295 (mPEG2000) 318.4 ± 3.9 0.004 �2.60 ± 0.1410 (mPEG2000) 268.6 ± 5.0 0.005 �1.18 ± 0.232 (mPEG5000) 213.2 ± 3.3 0.120 �4.02 ± 0.305 (mPEG5000) 256.3 ± 8.6 0.082 �3.28 ± 0.5010 (mPEG5000) 225.0 ± 1.6 0.007 0.73 ± 0.152 (mPEG5000) (pH 2) 370.8 ± 20.7 0.100 �13.50 ± 0.012 (mPEG5000) (pH 3) 454.9 ± 33.4 0.143 �19.93 ± 0.43

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be an excess of H3O+ that was supposed rapidly to end upthe propagation step of the polymerization and someresiduals of monomers would be left in the system. Onthe contrary, a higher pH value would lead to more OH�

dissociated from water, which would rapidly initiate thepolymerization instead of mPEG. Besides, too fast reactionwas likely to form aggregation of polymer. Consequently, itwas concluded that mPEG–PBCA nanocapsules could beproduced only at pH < 2.

3.6. Encapsulation efficiencies of nanocapsules

Fig. 7 illustrated the drug encapsulation efficiencies ofPEG–PBCA nanocapsules and PBCA nanoparticles. The

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encapsulation efficiencies of PEG–PBCA nanocapsules pre-pared in miniemulsion system were significantly higherthan those of PBCA nanoparticles. It seemed that the mini-emulsion system was preferable to the conventional emul-sion method in loading hydrophobic drugs, because its oilcore favored encapsulation of the drugs, rather than simplyadsorption at the surface of the particles. Additionally, theresults demonstrated that the encapsulation efficiencies ofthe nanocapsules prepared in miniemulsion rose dramati-cally when the amount of mPEG fed increased. It could beassigned to the important role of the mPEG molecules inthe preparation. The mPEG acted as the initiator of thepolymerization of butylcyanoacrylates as well as the stabi-lizer of the miniemulsion system. With the increase of themPEG in the suspension, the efficiency of polymerizationwas enhanced and larger amount of nanocapsules wereformed, thus the loading capacity could be improved. ThePEG–PBCA nanocapsules prepared using ethyl acetate asoil core also exhibited the similar tendency (data notshown).

3.7. Biocompatibility studies of nanocapsules

3.7.1. Hemolytic potential of nanocapsulesFig. 8 showed the hemolytic potential of PBCA nanopar-

ticles, 2% PEG–PBCA, 5% PEG–PBCA and 10% PEG–PBCAnanocapsules. It could be found that PEG modificationhas significantly decreased the hemolysis of the red bloodcells (RBCs). The PBCA nanocapsules at high concentration(2 mg/ml) exhibited a hemolysis rate of 19.8%, while thehemolysis rate of 2% PEG–PBCA with the same concentra-tion was 8.9%. And the hemolysis rate of 5% PEG–PBCAand 10% PEG–PBCA nanocapsules were 2.0% and 4.7%,respectively. The membrane of RBCs is soft and is com-

Fig. 7. Encapsulation efficiencies of PEG–PBCA nanocapsules and PBCAnanoparticles (Mean ± SD, n = 3).

Table 2Cell viability of QGY 7703 cells incubated with PEG–PBCA nanocapsule and PBCA

Sample 0.2 mg/ml

PBCA 27.57% ± 4.20%2% PEG–PBCA 88.19% ± 6.15%5% PEG–PBCA 77.97% ± 9.07%10% PEG–PBCA 91.74% ± 10.53%

posed of two-sublayers, the outer sublayer of which is neg-atively charged and the inner one is positively charged. Theinteraction between synthetic polymer and biological cellsurfaces related to the softness of the polymer surfacesas well as its surface charges. Through PEGylation, the sur-face charge density of the membranes decreases and thesurface becomes softer [21]. With the increase of the PEGamounts in the medium, the hemolysis rate decreased tillthe PEG content attached to (physically or chemically)the surface of the nanocapsules reached saturation. Buttoo much PEG amount that derived from high PEGylationmight change the osmotic pressure of the solution [22]and cause some extent of hemolysis. This is probably thereason that the hemolysis rate of 10% PEG–PBCA was high-er than that of 5% PEG–PBCA (see Fig. 8).

3.7.2. Cytotoxicity measurements (MTT assay)To evaluate the cytotoxicity of the polymers, the MTT

assay was performed to measure the viability of theQGY7703 cells incubated in medium containing 2%PEG5000–PBCA, 5% PEG5000–PBCA, 10% PEG5000–PBCAand PBCA nanoparticles. As illustrated in Table 2, therewas a significant increase (p < 0.05) in viability when thecells are in contact with the PEG–PBCA nanocapsules com-pared with the PBCA nanoparticles at various concentra-tions. This result suggested that PEG modification on thesurface could reduce the cytotoxicity significantly. As re-ported previously, nanoparticles could cause cytotoxicityby adherence of the particles to the cell membrane, degra-dation of the adhered nanoparticles and subsequent re-lease of cytotoxic degradation products [23]. Therefore,the improvement of cell viability in PEG–PBCA nanocap-sules may be attributed to the steric repulsion effect and

Fig. 8. Hemolytic effects of PEG–PBCA and PBCA polymers on dog eryt-hrocytes at concentration of 2 mg/ml and 1 mg/ml, respectively (Mea-n ± SD, n = 3).

nanoparticles (n = 6)

0.5 mg/ml 1.0 mg/ml

17.11% ± 3.65% 9.78% ± 1.64%52.74% ± 15.65% 43.64% ± 4.40%61.95% ± 6.19% 48.35% ± 1.25%47.58% ± 8.98% 30.29% ± 10.58%

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the biocompatible PEG coating outside the capsules, whichcould prevent the cell membrane from contacting the toxiccomponents directly. In addition, all of the four kinds ofsamples demonstrated a dose-dependent increase in cellviability. At the concentration of 0.2 mg/ml, there was nosignificant difference (p > 0.05) between 10% PEG5000–PBCA nanocapsules and the negative control. Therefore,the PEG–PBCA nanocapsules were considered to becytocompatible when the concentration was below0.2 mg/ml.

Y

4. Conclusion

In the present study, the PEG–PBCA nanocapsules wereprepared with the miniemulsion technique. The obtainednanocapsules showed a structure consisting of an oil coreand a PEG–PBCA outer layer, which greatly benefited theloading of hydrophobic drug. The hemolysis assay andthe MTT assay indicated that the PEG–PBCA nanocapsulespossessed fine biocompatibility and could be used as apromising carrier for hydrophobic agents.

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