Journal of Colloid and Interface Science 353 (2011) 363–371

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Journal of Colloid and Interface Science

www.elsevier .com/locate / jc is

In vivo NIR imaging with CdTe/CdSe quantum dots entrapped in PLGA nanospheres

Jin Soo Kim a,1, Kwang Jae Cho b,1, Thanh Huyen Tran c, Md. Nurunnabi a, Tae Hyun Moon d, Suk Min Hong e,Yong-kyu Lee a,⇑a Department of Chemical and Biological Engineering, Chungju National University, Chungbuk 380-702, Republic of Koreab Department of Otolaryngology, Head & Neck Surgery, The Catholic University of Korea, College of Medicine Uijeongbu St. Mary’s Hospital, Kyunggi-Do 480-717, Republic of Koreac Department of Applied Chemistry & Biological Engineering, Chungnam National University, 220, Gung-dong, Yuseng-gu, Daejeon 305-764, Republic of Koread Mediplex Corp., Seoul 135-729, Republic of Koreae Nano/Bio Chemistry Laboratory, Institute Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea

a r t i c l e i n f o

Article history:Received 8 May 2010Accepted 23 August 2010Available online 18 October 2010

Keywords:Quantum dotsPLGA nanospheresImagingTumorDetection

0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.08.053

⇑ Corresponding author. Fax: +82 043 841 5220.E-mail address: leeyk@chungju.ac.kr (Y.-k. Lee).

1 These authors are equally contributed to this artic

a b s t r a c t

Luminescent near-infrared (NIR) CdTe/CdSe QDs were synthesized and encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanospheres to prepare stable and biocompatible QDs-loaded nanospheres forin vivo imaging. QDs were encapsulated with PLGA nanospheres by a solid dispersion method and opti-mized to have high fluorescence intensity for in vivo imaging detection. The resultant QDs-loaded PLGAnanospheres were characterized by various analytical techniques such as UV–Vis measurement, dynamiclight scattering (DLS), fluorescence spectroscopy, and transmission electron microscopy (TEM). Finally,we evaluated toxicity and body distribution of QDs loaded in PLGA nanospheres in vitro and in vivo,respectively. From the results, the QDs loaded in PLGA nanospheres were spherical and showed a diam-eter range of 135.0–162.3 nm in size. The QD nanospheres increased their stability against photooxida-tion and photobleaching, which have the high potential for applications in biomedical imaging. Wehave also attained non-invasive in vivo imaging with light photons, representing an intriguing avenuefor obtaining biological information by the use of NIR light.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Quantum dots (nanometer-scale semiconductor nanocrystals,QDs) have attracted significant attentions during the last decadesbecause they can dramatically improve the use of fluorescentmarkers in biological imaging [1–3]. Due to their unique character-istics such as tunable fluorescence wavelength by size, sharp andsymmetrical fluorescence peak, strong and stable emission, highquantum yield, and broad excitation spectra, the QDs can providedistinct advantages over conventional organic dyes in vitro andin vivo [4–6].

However, these QDs are hydrophobic and so usually aggregatedin aqueous media. Their luminescences are easily impaired and theQDs are targeted by the mononuclear phagocyte system (MPS) [7].For more extensive and effective biological applications, QDs havebeen encapsulated or surface modified to prevent aggregation andmake them biocompatible. The various strategies have been usedto make them water-soluble, such as surface functionalizationwith water-soluble ligands [8–10], silanization [11], and encapsu-lation within block-copolymer micelles [12–14]. The strategy of

ll rights reserved.

le as co-first authors.

using polymers is generally superior to the surface modification,because: (a) there is no direct interaction with the QD surfaceatoms and it therefore can preserve the original quantum effi-ciency to a highest extent; (b) the presence of hydrophobic poly-mer domains around QDs may strengthen the hydrophobicinteraction to form more steady structures and consequently morestable and water-soluble QDs; and (c) these polymers can be tailor-made to have good stability in aqueous media and other functionalmoieties can be introduced on their surface. Polymeric encapsula-tion has been extensively studied for solubilization of hydrophobicdrugs and bioactive agents due to its unique properties thatinclude a nano-scaled size, high water solubility, high structuralstability, high carrying capacity of hydrophobic agents, and easi-ness in introducing functional moieties on the outer shell [15–17]. Poly(lactic-co-glycolic acid) (PLGA), in particular, is the mostfrequently used system because of its biocompatibility and biode-gradability [18,19]. In this study, luminescent Near IR QDs weresynthesized and encapsulated in biodegradable PLGA nanospheresfor the preparation of stable and biocompatible QDs-loadednanospheres. The nanospheres were characterized by UV–Vis,DLS, fluorescence spectroscopy, and transmission electron micros-copy (TEM). The stable and biocompatible QDs-loaded PLGAnanospheres provided sufficient circulation times and non-toxicimaging agents in vivo.

364 J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371

2. Materials and methods

2.1. Materials

PLGA (50:50, MW: 40,000–70,000), polyvinyl alcohol (PVA),cadmium oxide, cadmium chloride tech grade, tellurium powder200 mesh, selenium powder, n-tetradecyl phosphonic acid (TDPA),1-octadecene (ODE), trioctylamine (TOA), trioctylphosphine oxide(TOPO), dichloromethane (DCM), trioctylphosphine (TOP), hexa-decylamine (HDA), were purchased from Sigma–Aldrich (St. Louis,MO). The other chemicals and solvents were of reagent grade andused as received without further purification. SK-BR-3 cells(human breast cancer cells) and KB cells (human epidermoid carci-noma cells) were purchased from Korean Cell Line Bank (Seoul,Korea). Penicillin–streptomycin, fetal bovine serum (FBS), 0.25%(w/v) trypsin-0.03% (w/v) EDTA solution, RPMI-1640 mediumwas obtained from Invitrogen (Carlsbad, CA).

2.2. Synthesis of NIR QDs

To obtain the CdTe/CdSe QDs, a mixture of CdO (0.10 mmol),ODPA (0.205 mmol), and TOA (10 ml) was heated to 300 �C to geta clear solution. A solution of tellurium (0.2 mmol, dissolved in0.475 g of TOP) was quickly injected into this hot solution, andthe reaction mixture was allowed to cool to 250 �C for the growthof CdTe nanocrystals. Aliquots were taken out at different reactiontimes to monitor the reaction process by measuring the UV–Visabsorbance. The reaction was stopped when the desired nanocrys-tal size was reached. The synthesis was carried out under argongas. To avoid oxidation, the aliquots taken for the measurementswere added into anhydrous chloroform and stored under argongas before purification and measurements. For CdTe/CdSe core/shell, the CdTe QDs (0.02 g) were dispersed in TOPO (2 g) andHAD (2 g) before being heated to 190 �C. In addition, CdCl2 (0.1 g)was dissolved in TOP (3 ml) with gentle heating. After being cooledto room temperature, the resulting 0.2 M solution was mixed withSe (2.5 ml, 0.2 M in TOP). With a syringe pump, this mixture wasinjected within 30 min into the reaction flask containing the corenanocrystals at 190 �C. The crystals were then annealed at 170 �Cfor an additional 30 min. Core/shell nanospheres of various sizeswere obtained by adjusting the concentrations of CdCl2 and Se inTOP as well as the corresponding injection periods. The preparedCdTe/CdSe QDs were further purified by centrifugation andrepeated precipitation from methanol.

2.3. NIR QDs loading in PLGA nanospheres

QDs-loaded PLGA nanospheres were prepared by the followingmethod. In brief, 150 mg of PLGA was dissolved in 30 ml of DCMand different amounts of QDs in chloroform (5, 20, 40, 160 lM)were mixed together with moderate magnetic stirring for 1 h.The solution was homogenized at 24,000 rpm for 10 min after dis-persing in 50 ml of water containing PVA. Subsequent evaporationof dichloromethane was carried out with mechanical stirring for24 h at room temperature. The formed nanospheres were collectedby centrifugation and washed by dispersion in water and subse-quent centrifugation (12,000 rpm, 15 min). This final step wasrepeated three times in order to separate the QDs-loaded PLGAnanospheres from non-associated QDs. The loading amount ofQDs in PLGA nanospheres was controlled by the feed amount ofQDs from 5 to 160 lM. Finally, the particles were frozen-driedand obtained as a powder type. The yield of QDs-loaded PLGAwas around 90%. The encapsulation efficiencies were calculatedas follows.

Encapsulation efficiencies = [Amount of QDs encapsulated inPLGA nanospheres (g)] � 100/[Total QDs input (g)].

2.4. Measurement of size, morphology and zeta-potentials

The size of QDs-loaded PLGA nanospheres was measured by DLS(Otsuka, Japan). The morphology of the encapsulated QDs in PLGAnanospheres was examined using a JEM-100CX TEM (JEOL, Japan)and scanning electron microscopy (SEM) (JEOL, Japan). For TEMobservation, one drop of encapsulated QDs in PLGA was mountedon a thin copper grid. After light staining, the dried sample was ob-served under high vacuum. The SEM samples were prepared bysuspending 1 mg of nanospheres in 1 mL of water. Then, 200 lLof the suspended nanospheres were pipetted onto a stub,dehydrated, sputter coated with platinum, and examined at anaccelerating voltage of 25 kV. The average size, size distribution,and zeta-potentials of the QDs and QDs entrapped in PLGA nano-spheres were estimated by Zeta Sizer 3000 (Malvern Instruments,Worcestershire, UK). Samples were diluted with ultra pure waterfor zeta-potential measurement and 0.01 mol/L sodium chloridesolution to adjust the conductivity to 50 lS/cm for zeta-potentialmeasurement. The pH of the solution was around 6.5.

2.5. MTT assay and cellular uptake of QDs-loaded PLGA nanospheres

MTT assay was performed on KB cells by incubating at 37 �C for1 day with different concentrations of each compound in quadru-plicate. The control was incubated at 37 �C for 1 day without add-ing any drugs. This assay is based on the reduction of the yellowtetrazolium component (MTT) to an insoluble purple-colored for-mazan produced by the mitochondria of viable cells. After a 48 hincubation, 100 lL of medium containing 20 lL of MTT solutionwas added to each well and the plate was incubated for an addi-tional 4 h, followed by the addition of 100 ll of MTT solubilizationsolution (10% Triton X-100 plus 0.1 N HCl in anhydrous isopropa-nol, Sigma, Milwaukee, WI) to each well. The solution was gentlymixed to dissolve the MTT formazan crystals. The absorbance ofeach well was read with a microplate reader at a wavelength of570 nm. The background absorbance of well plates at 690 nmwas measured and subtracted from the 570 nm measurement.The results are expressed as % cell viability, obtained by dividingthe optical density values (OD) of the treated groups (T) by theOD of the controls (C) ([T/C � 100%]). In order to determine the up-take of the QDs-loaded nanospheres in cancer cells, human epider-moid and breast cancer cells (KB and SK-BR-3) were exposed toQDs entrapped in nanospheres and visualized using confocal lasermicroscopy (Carl zeiss, LSM 510, Germany). The KB cells or SK-BR-3 cells were grown on a Lab-Tek� II chamber slide (Nalge Nunc,Napevillem, IL). The concentrations of QDs were controlled to 5,20 and 40 lM. After 1 h incubation, the medium containing thecomplexes was aspirated from the wells. The cells were thenwashed three times with PBS buffer (pH 7.4) and finally 200 lLof 4% formaldehyde in phosphate buffer saline was added. Thesamples were observed as quickly as possible. Initial scanning ofthe cells was done under low power magnification (10�) and thenincreased up to 100�. The experiment was done at least threetimes.

2.6. In vivo imaging analysis

SKH1 mice (Orient Bio INC., Korea) were anesthetized with ket-amine (87 mg/kg, Virbac Laboratories, France) and xylazine(13 mg/kg, Kepro B.V., Netherland) via intraperitoneal injection.The NIR QDs-loaded nanoparticles (1 mg/mL in PBS) were injectedintravenously via the lateral tail vein. In vivo mouse images wereacquired with a time-domain diffuse optical tomography system.

J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371 365

Briefly, the animals were positioned on an imaging platform.Images were acquired at 1, 24, 48 and 72 h post-injection. The3D scanning region of interest was selected using bottom-viewCCD. All image analyses were performed using the Kodak in vivoimaging system (4000MN PRO, Kodak, USA). To obtain imagingsensitivity of QDs-loaded PLGA nanospheres in mice, different con-centrations of nanospheres were injected subcutaneously at fouradjacent locations on a mouse. All experiments were approvedby Institutional guidelines of the Institutional Animal Care andUse Committee (IACUC) of the Catholic University of Korea Collegeof Medicine in accordance with the NIH Guidelines.

2.7. Statistics

Statistical analysis was done using ANOVA. p < 0.01 was ac-cepted as statistically significant. Error bars represent standarddeviation.

3. Results and discussion

3.1. Characterization of NIR QDs

High quality NIR emitting CdTe/CdSe QDs were synthesized byinjecting a mixture of dissolved selenium and tellurium into a hotcoordinating solvent of trioctylphosphine oxide (TOPO) containing

Fig. 1. Schematic illustration of NIR QDs for in vivo imaging. (a) Structure of CdTe/CdSeQDs.

cadmium chloride. The QDs were composed of a CdTe/CdSe core/shell with emission wavelengths tuned from 550 to 800 nm.Fig. 1a illustrates a schematic representation of CdTe/CdSe QDsand PLGA structure. To prepare these QDs with the desired emis-sion wavelengths (emission peak: 760 nm) and optical propertiesas shown in Fig. 1b, we controlled the injection time interval withdifferent CdCl2 concentrations. The formation of the QDs was read-ily apparent by the change in the color of the reaction mixture fromclear to dark red and the shift of the emission toward longer wave-lengths. The photoluminescence (PL) properties of the QDs, includ-ing quantum efficiency, the peak position, and the PL full width athalf maximum of the emission spectrum (FWHM, 125 nm), did notshow any detectable change upon aging in air for several months.Occasionally, when the QDs precipitated in the solution and driedunder vacuum was kept for months, it took several hours to redis-perse the solid QDs completely in the solvents such as CHCl3. How-ever, by pouring the additional CHCl3 into the solution of QDs ordoing ultrasonication, the redispersion of the QDs from their con-centrated solutions could be performed without damaging theoptical properties of the QDs. Fluorescence quantum yield (QY)was determined by comparing their integrated emission of theQDs in chloroform solutions to that of the organic fluorophore rho-damine 6G dye in methanol solutions with equal optical density atthe excitation wavelength. The QY values were corrected for thedifferences in refractive index between chloroform and methanol.

QDs. (b) Fluorescence intensity and wavelength of NIR QDs. (c) TEM images of NIR

366 J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371

The QY of the QDs can reach as high as 0.5, which is much higherthan some other NIR emitting QDs synthesized in aqueous phase[20]. Fig. 1c provides the TEM image of CdTe/CdSe QDs. The indi-vidual CdTe/CdSe QDs was uniformly dispersed in the range of 3and 7 nm in diameter. The magnified Fig. 1c illustrated the repre-sentative of small particle size with spherical morphology. Previ-ous studies showed that QDs were more stable againstphotobleaching than NIR emitting organic fluorophores (Cy5.5)[21]. Also, the fluorescence spectrum of the QDs is symmetricand narrow and does not contain a front or tail compared withCy5.5 dye [21,22]. We selected QDs in the 650–850 nm emissionrange to prove the value of using NIR emitting QDs for biologicalimaging application.

3.2. NIR QDs-loaded PLGA nanospheres

To use NIR QDs as a targeting imaging agent for cell and animalimaging, QDs were entrapped into the PLGA nanospheres. To thebest of our knowledge, no reports are available on using Type IIQDs CdTe/CdSe-loaded PLGA nanospheres for in vivo imagingapplication. Before loading QDs within PLGA nanospheres, the con-centration of PVA was optimized to obtain stable nanospheres. Themean particle size and zeta-potentials of PLGA nanospheres andQDs-loaded PLGA nanospheres with different concentrations ofPVA and QDs were summarized in Table 1.

In the presence of 1% PVA concentration, the mean particle sizeof empty PLGA nanospheres was 230.8 ± 29 nm using DLS method.Increasing PVA concentration to 3%, the particle size of the PLGAnanospheres was decreased to about 137 nm. Further increasingPVA concentration to 5% and 10% of PVA concentrations resultedin slight decrease in the particle size of the nanospheres to about132 and to 125 nm. In the presence of 3% PVA the stability of PLGAnanospheres during lyophilization were preserved with negligibledifference in the mean particle sizes before and after lyophiliza-tion. Therefore, the PLGA nanospheres prepared with 3% PVA wasselected for QD loading. The QDs-loaded PLGA nanospheres pre-pared by solvent evaporation method were ca. 135–162 nm in sizedepending on the feed amount of QDs, which is insignificantly dif-ferent from these of the empty PLGA nanospheres (3% PVA). Thissize range falls within the accepted window for efficient cellularuptake and EPR (Enhanced Permeability and Retention) effect[16]. The QDs encapsulation efficiency was above 98% in all con-centration of QDs in the nanospheres, which is presumably dueto the good miscibility of QDs in the polymer matrix. The PLGAnanospheres had a negative zeta-potential ranging from �27.1 to�31.3 mV, indicative of the presence of carboxyl groups of PLGAon their surfaces as shown in previous study [23]. The shell ofCdTe/CdSe QD coated with capping ligand TOPO/TOP presentscomplex functional groups, including alkyl chain, phosphate, sul-fur, and oxide groups. The hydrophobic interaction between CdSeof QDs and alkyl group of TOPO could maintain the stable complexin organic solvents such as chloroform and toluene. After loading

Table 1Size distribution and zeta-potentials of PLGA and QDs-loaded PLGA nanospheres(n = 3, p < 0.001).

Samples PVA(%)

QDs(lM)

Average size(nm)

Zeta-potential(mV)

Shape

PLGA 1 – 230.8 ± 29 �31.3 Spherical3 – 135.7 ± 0.5 �27.1 Spherical5 – 132.6 ± 5.9 �28.4 Spherical

10 – 125.4 ± 9.3 �28.0 Spherical3 5 135.0 ± 20.3 �24.8 Spherical3 20 140.9 ± 18.5 �19.2 Spherical3 40 143.5 ± 10.5 12.3 Spherical3 160 162.3 ± 31.2 21.1 Spherical

with QDs, the zeta-potential values of QDs-loaded PLGA nano-spheres gradually increased to positive values depending on themolarity of Loaded QDs, showing the value of �24 mV and21.1 mV at 5 and 160 lM QD, respectively. Our results suggest thatthe positive charge of the QDs counterbalanced the negative chargeof the PLGA nanospheres, and the magnitude of the positive chargeof QDs loaded in PLGA nanospheres was affected by the amount ofthe encapsulated QDs. Lee et al. also reported the zeta-potential ofmicelles was changed to the positive charged surface after loadingQDs [24]. The surface characteristics of nanoparticles are an impor-tant factor determining their life span and fate during circulationrelating to their capture by macrophages [25]. Well-designednanoparticles should have a surface charge either slightly positiveor slightly negative for long-term circulation [26]. Thus, the surfacecharge of QDs-loaded PLGA nanospheres with 20 and 40 lM QDsare more suitable for in vivo imaging than that of the nanosphereswith 5 and 160 lM QDs.

Figs. 2 and 3 show the morphology of PLGA nanospheres pre-pared by changing the amount of PVA from 1% to 10% and QDs-loaded PLGA nanospheres (3% PVA) with varied feed amounts ofQDs, respectively. The nanoparticles appear as uniformly sphericalshapes before and after QDs loading indicating that the size andmorphology of the PLGA nanospheres were negligibly alteredaccording to loading amounts of QDs.

The optical characteristics of the QDs-loaded PLGA nanosphereswere examined using absorption and photoluminescence. Fig. 1bshows no shift of the absorbance peak of the QDs-loaded PLGAnanospheres compared to that of free QDs in chloroform. The pho-toluminescence emission peak at 760 nm of the QDs-loaded PLGAnanospheres is close to that of the QDs in chloroform. The QY of theQDs-loaded PLGA nanospheres was similar with that of the originalQDs (0.52). It was reported that a QY of 20% was sufficient forwhole body animal imaging [27]. Thus, the QDs-loaded PLGA nan-ospheres with a high QY might be useful for biological application.

Fig. 4 shows the internal structures of the PLGA nanospheresand QDs-loaded PLGA nanospheres with TEM. It further demon-strates that numerous QDs were encapsulated into each nano-sphere and that most of them were located on the inside of thenanospheres.

Fig. 4b showed the increased thickness of PLGA nanospheresafter loading QDs. Our data indicate that the increased thicknesswas mainly the result of strong hydrophobic interaction betweenthe PLGA fragment and TOPO of QDs. The fluorescence quenchingdue to hydrophobic interactions of QDs within the aqueous phasehas been a major problem in the biological application of QDs. Ourstrategy was to distribute the QDs efficiently in the hydrophobiccore by the controlling loading amount of QDs in the nanospheres.Furthermore, the QDs were chemically coated with hydrophobicTOPO agents, which maintained their stability in water for longerthan naked QDs during the encapsulation process.

To investigate the stability of QDs-loaded PLGA nanosphereswithin aqueous solution and in serum, we measured any changesin fluorescent intensity with time as shown in Fig. 5. First, the sta-bility of the QDs loaded in PLGA nanosphere were studied at fivedifferent pHs (5, 6, 7, 8 and 9) in 0.1 M PBS buffer (Fig. 5b).

The QDs-loaded PLGA nanospheres were dispersed into each3 mg/mL pH buffer solution separately for 2 h, the fluorescentintensity of the nanospheres was then measured using fluores-cence spectrometer (FluoroMate FS-2, Scinco CO., Ltd., Korea).The stability of the QDs-loaded PLGA nanospheres in the presenceof serum (5% (v/v) in PBS, FBS) was also measured to evaluate non-specific binding with proteins (Fig. 5c). To minimize interferenceby large molecules in FBS, the serum solution was filtered by usinga 0.2 lm filter membrane. The QDs-loaded PLGA nanospheres wereincubated in the FBS solution for 72 h and the fluorescent intensitychange of the nanospheres was monitored. Our results

Fig. 2. Scanning electron micrographs showing size distribution changes of PLGA nanospheres by different amounts of PVA: (a) 1%, (b) 3%, (c) 5% and (d) 10%.

Fig. 3. Scanning electron micrographs of PLGA nanospheres (PVA: 3%) after loading: (a) 5 lM, (b) 20 lM, (c) 40 lM, (d) 160 lM of QDs, respectively.

J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371 367

demonstrated that the pH change exerted a minimal effect on thestability of PLGA. However, the fluorescent intensity of the QDs-

loaded PLGA nanospheres after 1 day incubation in the bufferwas decreased to 79% of original value and then maintained 70%

(a)

(b)

200 nm 20 nm

200 nm 20 nm

Fig. 4. TEM observation of: (a) PLGA nanospheres stained with 2% phosphotungstic acid and (b) QDs loaded in PLGA nanospheres. The concentration of QDs was controlled to5 lM.

pH

Fluo

resc

ent i

nten

sity

(%)

0

20

40

60

80

100

120

Time (day)

Fluo

resc

ent i

nten

sity

(%)

0

20

40

60

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Time (hr)5 6 7 8 90 1 3 5 7 1 24 48 72

Fluo

resc

ent i

nten

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(%)

0

20

40

60

80

100

120

140(a) (b) (c)

Fig. 5. Stabilities of QDs-loaded PLGA nanospheres (n = 3, p < 0.001) at: (a) PBS buffer, (b) different pH and (c) 5% serum condition.

368 J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371

for 7 days as shown in Fig. 5a. We believed that the QDs-loadedPLGA nanospheres had enough high fluorescent intensity to be de-tected in the in vivo imaging. In 5% serum condition, the fluores-cent intensity of QDs-loaded PLGA nanospheres was a littleincreased up to 120% of the initial QDs after 24 h incubation. Itmay be due to the dilution effect of aggregated QDs in the surfaceof PLGA nanospheres under serum condition. We need more exper-iments to prove the phenomenon around surface of PLGA nano-spheres. Furthermore, they retained their fluorescence andloading after 72 h incubation in 5% serum, indicating no severeleakage of QDs from the PLGA nanospheres. A little amount ofQDs may be released from PLGA for a long-term because of biode-gradable property of PLGA. We will study the toxicity of the leakedTOPO-coated QDs from the PLGA nanospheres in vivo as furtherwork. We expect that the entrapped QDs in TOPO and PLGA nano-spheres can be removed from the body without severe side effectsbefore degradation. These results strongly indicated the excellentsolubility and stability of the PLGA nanospheres, suggesting a roleas a probe for imaging in the biological systems.

3.3. Cytotoxicities

It was important to investigate the acute cytotoxicity of QDs-loaded PLGA nanospheres before applying them to cells for drugdelivery or imaging studies. The toxicities of the PLGA nanospheres

and QDs-loaded PLGA nanospheres were determined by perform-ing cytotoxicity tests using KB cells (5 � 103 cells/mL). The numberof cells that survived incubation was estimated using a MTT assay.Fig. 6 shows the cell viability after 24 and 48 h treatment withempty PLGA nanospheres and QDs-loaded PLGA nanospheres atdifferent amounts of Loaded QDs and the nanospheres concentra-tions. The viability of cells exposed to QDs-loaded PLGA nano-spheres was comparable with the viability of cells exposed toempty PLGA nanospheres (more than 60%) in the concentrationranged from 0.01 to 10 lg/mL for 24 and 48 h indicating that no se-vere cytotoxicity was observed when KB cells were treated withQDs-loaded PLGA nanospheres. Different amounts (5, 40, 100 and160 lM) of QDs in PLGA nanospheres were used to observe thetoxic effect of QDs on the cells in a concentration manner. TheKB cells presented above 60% cell viability regardless of the con-centrations of QDs loaded in PLGA nanospheres. The toxicity ofQDs not only depends on the concentration of free Cd2+ ions butalso on the ingestion of particles by a cell and where they arestored. These results indicate that the release of Cd2+ from QDsmay be reduced to a great extent by employing core/shell particlesor coating of the QDs with TOPO polymer and loading into PLGAnanospheres. These results are well in accordance with other re-search groups treated QDs with micelle or TOPO [1,12]. Further-more, very little QD release from the nanospheres is expectedover several days because of QD hydrophobicity. Thus, short term

Fig. 6. Cell viabilities of KB cells treated with PLGA nanospheres and QDs loaded in PLGA nanospheres at different concentrations (n = 3, p < 0.01) for: (a) 24 and (b) 48 hincubation, respectively. Concentrations of PLGA or QDs-loaded PLGA nanospheres were controlled to 0.01, 0.1, 1 and 10 lg/mL.

J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371 369

imaging studies would be performed without concerning for nano-particle-induced acute toxicity. In addition, according to statistics,the differences among the groups were not significant because p-values among the groups were greater than 0.05. Therefore, wefound that the effect of QDs-loaded PLGA nanospheres on cell via-bility and metabolic activities was not numerically significant.

3.4. Confocal laser scanning microscopic study

Whether the QDs-loaded PLGA nanospheres can be taken up bycells is an important consideration in diagnostics of diseases, espe-cially for cancer cell imaging and targeting by intratumoral injec-tion. This is because a higher uptake of the nanospheres bycancer cells may result in better molecular imaging outcomes.We examined the cellular uptake of QDs-loaded PLGA nanospheresusing two kinds of cancer cells, SK-BR-3 cells and KB cells. The celllines have different origin from human cancer cell and were usedto evaluate cellular uptake of the QDs-loaded nanospheres accord-ing to cell types. Fig. 7 shows CLSM images of SK-BR-3 and KB celllines after 1 h incubation with QDs-loaded PLGA nanospheres atdifferent concentrations of the QDs. The fluorescence images ofthe cells showed that red signals of the QDs-loaded nanosphereswas clearly observed regardless of cell types. From observation,the fluorescent intensities of QDs in the cells were increased withincreasing the concentration of QDs loaded in PLGA nanospheresfrom 5 to 20 and up to 40 lM. The cellular uptake of nanoparticleswas mainly controlled by particle size, surface charge and shape ofthe nanoparticles [26]. However, the particle size of the QDs-loaded PLGA nanospheres was insignificantly different at differentconcentrations of loaded QDs. Thus, the difference in the cellularuptake might be controlled by surface charge of the nanospheresin which the higher extent of cellular uptake was achieved withthe slightly positive charged nanospheres. More importantly, indi-vidual cell images clearly reveal that the QDs-loaded nanosphereswere internalized into cells and but localized in the cytoplasmcompartment rather than the nucleus because the fluorescentintensity of the nucleus was weaker than that of the other areas.This might be attributed to surface property and particle size ofthe nanospheres. It was reported that QD-loaded nanoparticleswere internalized into PC12 cells via nanoparticle endocytosis,particularly via tyrosine kinase A receptor in differentiated PC12cells [28]. In that study, the QDs modified with protein showedsmaller particle size than QD-loaded nanoparticles leading to high-er levels of nanoparticle endocytosis compared to the QD-loadednanoparticles. In the future, PLGA carboxyl groups may be

modified for surface conjugation of targeting ligands, which mayenhance the endocytosis and intracellular delivery of the QDs-loaded PLGA nanospheres.

3.5. Biodistribution of QD loaded PLGA nanospheres

A NIR whole animal imaging approach was used to investigatethe in vivo biodistribution of the QDs-loaded nanospheres inSKH1 mice. Fig. 8a–d gives the fluorescence signal and intensitydistribution as a function of time for the QDs-loaded PLGA nano-spheres delivered systemically via tail vein injections. Fig. 8eshows a concentration dependent NIR imaging using different con-centrations of QDs-loaded PLGA nanospheres. From the results,QDs loaded in PLGA could be observed clearly just 1 h post-injec-tion due to their high quantum yields and high absorbency. Forgood visualization through a significant thickness of tissue(>1 mm), stable NIR QDs should be necessary. The QDs-loaded nan-ospheres may enable visualization in whole animals at greaterdepth and with increased sensitivity. Stronger signals of the QDs-loaded PLGA nanospheres were visualized in various organs ofthe mouse after 24 h (Fig. 8b), indicating the stability of the nano-spheres in physiological environments. More importantly, thestrong signal from the fluorescent tissue distinguished it fromthe autofluorescence of the skin at low concentration.

The biocompatible PLGA nanospheres were still detected in theorgans for 48 h after i.v. injection of the nanospheres. The fluores-cence of QDs loaded in PLGA nanospheres were not observed fromthe body at 72 h after injection, assuming the concentration of QDsloaded in PLGA nanospheres was not enough to be detected or anextent of removal of QDs from the body. To prove the completeremoval of the QDs from the body, we need more experimentssuch as tissue evaluation as further study. Our preliminaryin vivo imaging study showed an advantage from the prolongedcirculation time afforded by the PLGA nanospheres.

The safety of QDs is one of the major concerns for in vivo QDs-based imaging techniques. A recent paper suggested that cytotoxic-ity of QDs, which is mainly induced by release of Cd2+ ions into thecellular environment, was greatly reduced by coating the bare QDswith polymers [29–31]. In the present study, QDs were entrappedin the core of polymeric nanospheres and did not show any leakageafter 72 h incubation in serum, or any loss of fluorescent intensity(Fig. 5c). From the MTT assay, we also confirmed that the cell viabil-ity of KB cells treated with PLGA nanosphere were similar with thatof KB cells treated with QDs loaded in PLGA nanospheres at 48 hincubation. Therefore, the toxicity of QDs in PLGA nanospheres

Fig. 7. CLSM observation of: (a) SK-BR-3 cells and (b) KB cells treated with QDs-loaded PLGA nanospheres. Each concentration of the QDs loaded in PLGA nanospheres wascontrolled to 5 lV, 20 lV and 40 lV.

Fig. 8. Body distribution and sensitivity of QDs-loaded PLGA nanospheres in mice. Whole animal imaging was observed after tail vein injection of 20 lM QDs-loaded PLGAnanospheres at: (a) 1 h, (b) 24 h, (c) 48 h and (d) 72 h. (e) Fluorescent intensities of QDs in mice according to concentration of QDs loaded in PLGA nanospheres 24 h post-injection (injection volume: 0.25 mL).

370 J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371

was considered to be low. The coating PLGA polymer is a well-devel-oped copolymer, and its safety has been well-acknowledged [32,33].Therefore, the application of QDs loaded in PLGA on cancer cells isbelieved to be safe. The application to the human body is still contro-versial and needs more studies on toxicity as further work. No

obvious side effects were observed in our animal experiments (datanot shown). However, since the safety of the probe is now one of themajor concerns for in vivo imaging, the long-term side effects of QDsloaded in PLGA still need to be considered very carefully and deservefurther research before their further application.

J.S. Kim et al. / Journal of Colloid and Interface Science 353 (2011) 363–371 371

The nanocrystal QDs offer numerous potential applications insingle cell and animal bioimaging. However, conventional QDs-based imaging often encounters poor stability in aqueous solutionsand biological systems, mainly due to photocatalytic destruction ofthe mercaptoacetic acid coating and precipitation of the nanocrys-tals [34–36]. Here, the use of TOPO-coated QDs encapsulation intoPLGA nanospheres enables their potential application in imagingin vivo. Our data clearly suggested that QDs were encapsulated intothe core of PLGA nanospheres with high loading efficiency withoutchanging the morphology, size and stability of nanospheres. Webelieve that this phenomenon is mainly due to the strong hydro-phobic interaction between TOPO and the PLGA fragment of thepolymer, which composed the core of the resulting PLGA nano-spheres [37,38]. In the course of primary emulsification, TOPO-QDs were positioned inside the PLGA nanospheres and then thesurface was solidified after solvent evaporation. The prepared QDloaded PLGA nanospheres showed higher solubility and stabilityin water compared to free QDs. The advantages of this preparationprocedure were obvious. By using it, we obtained nanosphereswith high payload capacity and improved stability in both waterand serum (Fig. 5). In addition, the photo-stability of the encapsu-lated QDs was significantly improved compared with that of freeQDs. In contrast, previous works in which QDs were encapsulatedin the chloroform/ethanol solvent mixture to obtain QDs-loadedmicelles had several drawbacks such as low payload (one QD permicelle) and easy aggregation [36]. Both PLGA and micelles havebeen used to improve biocompatibility of QDs in vitro and in vivo.Especially, micelles are very useful to observe cell imagingin vitro. However, micelles are not stable in vivo compared to lipo-some or nanospheres because it depends on critical micelle con-centration (CMC). At a low concentration, the structure ofmicelles is easily loosed and most of QDs from the micelles arereleased before reaching to target site in vivo.

We have demonstrated that QDs entrapped in PLGA nano-spheres have many desirable characteristics as imaging agentdelivery vehicles, including water solubility, low cytotoxicity, sta-bility, high cellular uptake and long circulation time in vivo. Theinternalization and long circulation time of the PLGA nanospheresincreases the retention time and amount of the nanoparticles in-side the cells. Both of these properties fit very well with the criteriaof a molecular target for tumor therapy and imaging. We have alsoshown that the QDs loaded in PLGA nanospheres are sensitiveenough to be detected inside the body at a low concentration.Therefore, our results demonstrate the potential applications ofQDs loaded in PLGA nanospheres for the detection of disease usingin vivo imaging method.

Acknowledgments

This research was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF)funded by the Ministry of Education, Science and Technology(2010-0021427) and was financially supported by the Ministry ofEducation, Science Technology (MEST) and Korea Industrial

Technology Foundation (KOTEF) through the Human ResourceTraining Project for Regional Innovation.

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