study of the selective uptake progress of aptamer-modified plga particles by liver cells

Full Paper

Study of the Selective Uptake Progress ofAptamer-Modified PLGA Particles byLiver Cellsa

Dahai Yu, Yuying Zhang, Zhengwei Mao,* Changyou Gao*

It is of paramount importance to study the cellular uptake processes of particles with definedsurface property, especially the uptake pathways and intracellular transportation. In thisstudy, aptamer AS1411 molecules, which are known to specifically bind the over-expressed
nucleolin on cancer cell membrane, were conjugatedonto bovine serum albumin-decorated poly(D,L-lactide-co-glycolide; PLGA, F400nm) particles with a density of1–1.7 molecule/10 nm2. The aptamer-modified PLGAparticles were preferably ingested by liver cancer cellswith higher amount and faster rate. The clathrin-mediated endocytosis and macropinocytosis pathwaysplayed a more important role in uptake of the aptamermodified particles.
D. Yu, Y. Zhang, Z. Mao, C. GaoMOE Key Laboratory of Macromolecular Synthesis andFunctionalization, Department of Polymer Science andEngineering, Zhejiang University, Hangzhou 310027, ChinaE-mail: [email protected]. GaoState Key Laboratory of Diagnosis and Treatment for InfectiousDiseases, First Affiliated Hospital, College of Medicine, ZhejiangUniversity, Hangzhou 310003, China,E-mail: [email protected]

aSupporting Information is available from theWiley Online Library orfrom the author.

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Biosci. 2013, 13, 1413–1421

wileyonlinelibrary.com

[8–11] [12–15]
1. Introduction
In order to decrease the dosage and subsequent toxicity,

several active and passive strategies have been developed

toenhance thedrugconcentration indesiredcells or tissues,

amongwhich decoration of the particle surfacewith ligand

molecules for specific cells or tissues has attracted

tremendous attention in recent years.[1–4] The folate,[5–7]

Arg-Gly-Asp (RGD), transferrin, and monoclonal

antibody[16–19] are the typical ligands used to target

particles to many kinds of carcinomas.

Due to their particular small size and/or other function-

alities, the particles are possibly internalized into cells

when they come into contact with the cells. In the

endocytosis process the particles are firstly attached to

the cell membrane through specific or nonspecific inter-

actions, and then are wrapped by cell membrane to form

membrane-bounded vesicles. The particles can enter cells

by several different endocytic pathways, such as phagocy-

tosis, clathrin-mediated endocytosis, caveolae-mediated

endocytosis, macropinocytosis, and clathrin- and caveolin-

independent endocytosis.[20] The internalization pathways

not only influence the uptake efficiency but also the

intracellular fate of the particles.[21] Moreover, the release

of the drug into the enzymatic environment of the

lysosomes or directly in the cell cytoplasm will, indeed,

has different pharmacological activities.[22] Therefore, the

uptake pathways and intracellular transportation of

particles should be clarified with great care.

It is known that the cellular uptake process and

intracellular fate of the particles are dominated by their

DOI: 10.1002/mabi.201300165 1413

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D. Yu, Y. Zhang, Z. Mao, C. Gao

1414

surface chemistry especially the existence of ligand

molecules.[22] Generally, the surface-tethered ligand mol-

eculesare feasible foruptakeof theparticles.However, their

influence on theparticle uptakepathways and intracellular

location is still quite controversial. For example, Ferris et al.

observed that transferrin and cyclic-RGD peptides-modifi-

cation enhanced the adherence of mesoporous silica

particles to the cancer cell membrane, suggesting the

membrane receptor-mediated endocytosis.[23] Tung et al.

found that human lung carcinoma A549 cells endocyto-

sized more silica particles decorated with monoclonal

antibody against epidermal growth factor receptor (EGFR,

which is over-expressed on A549 cells) compared with

the unmodified ones. Both particles were internalized

by macropinocytosis and clathrin-mediated pathways,

and resided in lysosomes.[24] Fujiwara et al. found that

octaarginine-modified liposomes were internalized by

Madin-Darby canine kidney cells (MDCK cells) and located

in endosomes and lysosomes. By contrast, ligand-free

liposomes were always located in lysosomes.[25]

Mao et al.[26] demonstrated that Tat peptide decora-

tion affected the subcellular distribution of silica

particles, resulting in localization of the particles in the

cell nuclei.

Recently, aptamers,[27] which are oligonucleic acids or

peptides that bind to a type of specific target molecules,

have shown potential applications in diagnostics, purifica-

tion processes, drug discovery, and even therapeutic

approaches.[28,29] Previous studies have demonstrated

that functional particles with surface-tethered aptamers

have potential applications in tumor diagnosis and

therapy.[30–35] AS1411 is the first oligodeoxynucleotide

aptamer to reach phase I and II clinical trials for the

potential treatment of cancers, including acute myeloge-

nous leukemia.[36,37] It shows high binding affinity to

nucleolin, which is overexpressed on the plasma

membrane of tumor cells.[38–41] Therefore, covalent conju-

gation or physical immobilization of AS1411 onto

particles will largely enhance their targeting efficiency to

tumor cells. Nevertheless, so far most attention has been

paid to realizing either high selective targeting and anti-

tumor efficacy,[42–44] or bioimaging and tracking cancer

cells.[45–47] Little attention is focused on the uptake

processes. It is of high demand to clarify the cellular uptake

processes of the AS1411-modified particles, especially

the uptake pathways and intracellular transportation

because targeting therapeutics to individual intracellular

compartments has resulted in benefits to therapies

associated with these unique organelles.

Our previous study have shown that the surface

modification of PLGA particles with anti-foulingmolecules

bovine serum albumin (BSA) could significantly increase

their stability in physiological fluids and reduce the

adsorption of serum proteins and thereby the uptake by

Macromol. Biosci. 201

� 2013 WILEY-VCH Verlag GmbH

endothelial cells.[48,49] Taking this advantage, in this work

the PLGA–BSA particles were further covalently immobi-

lizedwith the aptamer AS1411molecules, and their uptake

by liver cancer cells (QGY-7703 cells) with over-expressed

nucleolin and normal liver cells (Hepli cells) was compared

in terms of uptake kinetics, pathways and subcellular

distributions. Herein, we investigated the cell–particle

interactions at molecular and subcellular levels, and

provided insight in design of functional particles for

selective intracellular delivery.

2. Experimental Section

2.1. Materials

Poly(D,L-lactide-co-glycolide (LA: GA¼75: 25,Mw ¼ 130 kD), BSA, 1-

ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride

(EDC), N-hydroxysuccinimide (NHS), amiloride–HCl, amanta-

dine–HCl, sodium azide, genistein, 40,6-diamidino-2-phenylindole

(DAPI), cytochalasin D (CytD), and 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphe-nyltetrazolium bromide (MTT) were purchased from Sigma-

Aldrich. Sodiumdodecyl sulfate (SDS)was purchased fromHaotian

Co., Ltd. (China). Anti-EEA-1 (early endosome marker) and

LysoTrackerGreenwereobtained fromInvitrogenCo., Ltd.Aptamer

AS1411-NH2 and Cy3—AS1411—NH2 were purchased from

Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals

were analytical grade andused as received.Milli-Qwaterwas used

throughout the experiments.

2.2. Particle Preparation

The PLGA–BSA (PB) particles were prepared by an O/W emulsion–

solvent evaporation method as described previously.[48] Briefly,

after 1mL 2w/v% PLGA dichloromethane solution (organic phase)

was added into 4mL 3% BSA solution (water phase), the mixture

solution was emulsified with an ultrasonicator (MISONIX Ultra-

sonic liquid Processors, 5W) for 20 s. The obtained emulsion was

poured into 150mL water, and stirred for 3 h at room temperature

with a magnetic stirrer until the organic solvent was completely

evaporated. The PB particles were collected by centrifugation at 12

500 rpm for 10min, and washed with water five times to remove

free BSA in the water phase and freeze-dried for storage. The PB

particles containing Nile red (NR) were similarly prepared by

adding 0.2mgmL�1 NR into the PLGA solution before mixing with

the BSA solution, respectively.

2.3. Surface Conjugation of Aptamer AS1411

The sequence of AS1411 is 50-(GGTGGTGGTGGTTGTGGTGGT-GGTGG). The AS1411 aptamer capped with an amino end group

(aptamer-NH2)was covalently conjugated to the PB particles using

carbodiimide chemistry. In brief, the 0.5mL suspension of 1mg

mL�1 PB particles was mixed with 1mg EDC and 0.5mg NHS to

activate the –COOH groups. 20min later, 0.5mL aptamer–NH2 (or

Cy3–aptamer–NH2) solution was added and the reaction was

maintained for 40min under shaking at room temperature. The

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Study of the Selective Uptake Progress of Aptamer-Modified PLGA Particles . . .

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PLGA–BSA–aptamer (PB-Apt) particles were collected by centrifu-

gation at 12 500 rpm for 10min, andwashedwithwater five times

under ultrasonication to remove physically adsorbed aptamer

molecules.

The fluorescence intensity of the aptamer solution before and

after reaction was compared to assess the grafting amount of the

aptamer by fluorescence spectroscopy (LS55, PerkinElmer, USA)

using Cy3–aptamer–NH2.

2.4. Particle Characterization

2.4.1. Morphology

Themorphology and the size of the PB and PB-Apt particles in a dry

state were analyzed by transmission electron microscopy (TEM,

Philips TECNAL-10). A drop of the particles suspension was added

onto a copper grid with a carbon membrane and dried at room

temperature. 100 particles were analyzed to provide statistical

result of the particle size.

2.4.2. Size and Surface Charge

The size and surface charge of the PB and PB-Apt particles in water

(pH 6.6) and DMEM/10% fetal bovine serum (pH 7.4) were

determined by dynamic light scattering with Beckman Delsa

Nano (Beckman Coulter), respectively.

2.5. Cell Uptake

2.5.1. Cell Culture

The immortalized human normal liver cells (Hepli cells) were

kindly donated by Dr. Jun Li from the First Affiliated Hospital,

College of Medicine, Zhejiang University. The human liver cancer

cells (QGY-7703 cells) were obtained from the Cell Bank of Typical

Culture Collection of Chinese Academy of Sciences (Shanghai,

China). The Hepli and QGY cells were maintained at 37 8C in a 5%

CO2 humidified environment in low glucose DMEM and high

glucose RPMI 1640 (Gibco, USA), respectively. Both culture

mediumswere supplementedwith 10%FBS, 100UmL�1 penicillin,

and 100mgmL�1 streptomycin.

2.5.2. Cellular Uptake of the PLGA Particles

The cellswere seeded on a 24-well plate at a density of 1� 105 cells

per well and allowed to attach for 16 h. Then they were incubated

with 50mgmL�1 of NR-labeled PLGA particles (PB and PB-Apt) for

different time, or with various concentrations of NR-labeled PLGA

particles for 12h. After washed five times with PBS to remove

the free particles, the cells were detached by trypsin. The uptake

amount of the NR-labeled PLGA particles was determined by flow

cytometry (FACS Calibur, BD).

2.5.3. Uptake Pathway

In order to clarify the role of ligand–receptor interactions in the

processof endocytosis, theQGY-7703cellswere incubatedwithNR-


� 2013 WILEY-VCH Verlag Gmwww.MaterialsViews.com

labeled PLGA particles for 2 h in the presence and absences of 3mL

100mM free AS1411 at 4 8C, respectively. The cellular uptake of thePLGA particles was then determined by flow cytometry. To

determine the uptake pathways, the cells were firstly incubated

at 4 8C andwith 100mM sodium azide in the presence of particles,

respectively. Different pharmacological inhibitors, including2mM

amiloride–HCl, 1�10�3M amantadine–HCl, 100�10�3

M genis-

tein, and10mgmL�1 cytochalasinDwerealsoused to treat the cells

for 1 h before incubation with the PLGA particles, respectively.

2.5.4. Intracellular Distribution

Fluorescent stainingof early endosomes, lysosomes, and cell nuclei

was performed to display the intracellular distribution of the PLGA

particles by confocal laser scanning microscopy (CLSM, TCS SP5,

Leica). Briefly, after incubatedwith 50mgmL�1 of NR-labeled PLGA

particles for desired time, the cells were carefullywashedwith PBS

three times, and continually culturedwith anti-EEA-1, LysoTracker

Green, and DAPI at 37 8C for another 30min, respectively.

2.6. Statistical Analysis

All values are expressed as mean� standard deviation (SD).

Statistically significant value is set as p <0.05 based on the

Student’s t-test.

3. Results and Discussion

3.1. Characterization of PLGA–BSA–Aptamer Particles

The PLGA–BSA particles were prepared via an O/W

emulsion-solvent evaporationmethodwith BSAmolecules

as stabilizers in the water phase. As a result, the obtained

PLGA particles had a shallow shell (several tens of nm) of

BSA molecules (Figure 1C), contributing about 6% of the

particle weight. These BSA molecules were very stable and

cannot be removed even under strong ultrasonication.[48]

This feature enabled the covalent tether of amino end-

capped AS1411 by the carboxyl groups of BSAmolecules in

the presence of EDC/NHS. To quantify the grafting amount,

Cy3 labeled aptamer molecules were used. Of the 2.0mg

feeding amount, 1.9mg aptamer molecules were conjugat-

ed onto 1mg PB particles (PB-Apt2), suggesting the very

high conjugation efficiency. Taking into account the

average particle size (Table 1), this value equaled to

3.8� 104 molecules per particle or 1 molecule/10 nm2.

When the feeding amount of aptamer was raised to 5mg

per mg PB particles, the grafting amount reached to 3.3mg

(PB-Apt3),which equaled to 6.3� 104molecules per particle

or 1.7 molecules/10 nm2.

As representatively shown in Figure 1 and Supporting

Information, Figure S1, both the PB and PB-Apt3 particles

possessed a size of 170nm in a dry state and a typical core–

shell structure regardlessof theconjugationofaptamer.The

size of PB-Apt3 particles (�430nm due to the hydration of

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Figure 1. A,B) SEM and C,D) TEM images of A,C) PLGA–BSA (PB)particles and B,D) PB-Apt3 (3.3mg aptamer per mg PB particle)particles, respectively.

www.mbs-journal.de


1416

surface protein layer and possible aggregation) kept

unchanged in comparison with that of the PB particles in

both water and serum-containing cell culture medium,

suggesting thegoodcolloidal stabilityof theparticlesdue to

the surface coating of BSA molecules. The PB particles

possessed negative charge on their surface due to the BSA

protecting layer, which was about �30mV in water and

�10mV in serum-containing medium.[48] The surface

charge property was not significantly altered after the

EDC/NHS treatment and conjugation of the AS1411

aptamer molecules (Table 1). Therefore, the potential

influence of physical parameters, such as size and surface,

charge on the cellular uptake of the aptamer-tethered PLGA

particles could be safely ruled out.

3.2. Cellular Uptake

The aptamer-tethered PLGA particles are expected to be

selectively delivered to the tumor cells, which could then

improve the therapeutic effects of the encapsulated

substances.[50] In this regard, the cellular internalization

Table 1. The size and surface charge properties of PB, PB-Apt2, and

Sample

Size [nm]

Water 1 640/1

PB particles 435� 13 438

PB-Apt2 particles 434� 26 441

PB-Apt3 particles 435� 27 441



of the nanoparticles is very important. In this study two

types of liver-derived cellswereused.QGY7703 cell is a type

of hepatic carcinoma cell that over expresses nucleolin (the

receptor of aptamerAS1411) on cellmembrane. Hepli cell is

a type of immortalized normal liver cells, which express a

normal level of nucleolin on cell membrane. By the

comparison, the differences in delivery and uptake of the

aptamer-modified particles can be distinguished. To make

the PLGA particles detectable via fluorescence microscopy

andflowcytometry, a hydrophobic dye, NR,was pre-loaded

during the particle fabrication. Due to its very poor

solubility in culture medium, the release of NR from the

particleswasneglectable, asdemonstratedbythe transwell

experiments.[48] In the FCMmeasurement, the logarithmic

fluorescence intensity of untreated cells was set between

100 and 101, and those cells with the intensity larger than

101 were considered as the positive ones (the cells ingested

the PLGA particles).[51] As shown in Figure 2A, the average

fluorescence intensity per cell increased along with the

prolongation of culture time for all types of the PLGA

particles andboth types of cells,with faster rates during the

first 8 h. In general, the normal cells (Hepli) internalized a

similar amount of PB and PB-Apt2 particles during the

whole experimental period (p< 0.05). However, the fluo-

rescence intensity of the Hepli cells incubated with the PB-

Apt3 particles was higher compared to that of the cells

incubatedwith the PBandPB-Apt2particles during thefirst

8 h, suggestingmorePB-Apt3particleswere takenupby the

Hepli cells. This difference disappeared at 12h. This might

be attributed to the fact that someamount of nucleolin also

existed on the surface of Hepli cells. Thus higher density of

aptamer AS1411 would lead to the enhanced recognition

and internalization of PB-Apt particles by Hepli cells to

some extent.

TheQGY-7703 cells took up a significantly larger amount

of PB-Apt2 particles than PB particles (p< 0.05) during the

whole experimental period, whichwas about four times at

the maximum at 8h. They internalized evenmore PB-Apt3

particles except at the first hour. During all the time points

(p< 0.05) except the first hour, the QGY-7703 cells also took

up significantly larger amounts of the PB-Apt2 and PB-Apt3

particles, which were 2 and 2.2 times to the Hepli cells at

12h, respectively.

PB-Apt3 particles.

Zeta potential [mV]

0% FBS Water 1 640/10% FBS

� 30 �34� 0.7 �17� 1.4

� 20 �35� 4.2 �16� 1.5

� 24 �36� 5.6 �15� 0.7

3, 13, 1413–1421


1h 4h 8h 12h0

100

200

300

400

Ave

rage

fluo

resc

ence

inte

nsity

per

cel

l

Time

PB@QGY PB-Apt2@QGY PB-Apt3@QGY PB@Hepli PB-Apt2@Hepli PB-Apt3@Hepli

0 20 40 60 80 100

0

200

400

600

800

*

*

**

Ave

rage

fluo

resc

ence

inte

nsity

per

cel

l

Concentration (µg/ml)

PB@QGY PB-Apt3@QGY PB@Hepli PB-Apt3@Hepli

(B)(A)

Figure 2. Uptake of the PLGA–BSA (PB), PB-Apt2 (1.9mg aptamer permg PB particle) and PB-Apt3 particles by QGY-7703 and A) Hepli cells as afunction of culture time with a particle concentration of 50mgmL�1, and B) particle concentration with a culture time of 12 h (B). Data weremeasured by flow cytometry and averaged to each cell. Asterisk indicates significant difference at p<0.05 level.


www.mbs-journal.de

To consolidate the above observation, the dose-depen-

dent uptake behaviors of PB and PB-Apt3 particles by these

two types of cells were investigated (Figure 2B). At a fixed

co-culture timeof12h, thePLGAparticleswere increasingly

internalized into or adsorbed onto the cells, almost linearly

alongwith the increase of particle concentration regardless

of the types of the particles and cells. However, the uptake

amount of PB-Apt3 particles by QGY-7703 cells was

significantly higher than that by Hepli cells for all the

tested concentrations (p< 0.05). Moreover, at each fixed

particles concentration, the QGY-7703 cells ingested

obviously higher amount of the PB-Apt3 particles than

that of the PB particles (p< 0.05). By contrast, theHepli cells

did not show obvious difference in the ingestion of the PB

and PB-Apt3 particles. These comparison results confirmed

that the aptamer-conjugated PLGA particles were selec-

tively delivered to the QGY-7703 cancer cells, which

expressed more nucleolin on the cell membrane for the

targeting interaction with the aptamer AS1411 ligand.[52]

As expected, this feature enhanced the therapeutic effect of

camptothecin loaded in the PB-Apt particles than loaded in

the PB particles (Supporting Informaiton, Figures S2,S3).

3.3. Uptake Pathway

The particles can enter cells by several different endocytic

pathways, such as phagocytosis and pinocytosis. Phagocy-

tosis is conducted primarily by specialized cells, including

macrophages,monocytes, and neutrophils, which can clear

out large particles in blood such as pathogens and debris of

dead cells. In contrast, pinocytosis can operate in all

mammalian cells. Clathrin-mediated, caveolin-mediated,

macropinocytosis, and clathrin- and caveolin-independent

endocytosis are four major processes of pinocytosis. These

mechanistically diverse and highly regulated endocytic

pathways could influence the intracellular fate of the



particles and cellular organismal homeostasis.[20,53,54]

Recent data also showed that the nucleolin on cell

membrane might act as a macrophage receptor,[55] and

AS1411 could be endocytosized by cells through macro-

pinocytosis stimulated by a nucleolin-dependent mecha-

nism.[56] As shown in Figure 3A and Supporting Informa-

tion, Figure S4, uptake of the PB particles by the QGY-7703

cells was almost completely inhibited at 4 8C (the low

temperature is beneficial to minimize the metabolism of

cell plasma membrane), proving the energy-dependent

nature of particle uptake. This nature was further verified

by the significant blocking of cellular uptake of the PLGA

particles regardless of their surface chemistries and cell

types after treatment with 100� 10�3M sodium azide

(Supporting Information, Figure S4). However, about 40%of

the uptake efficiency of the PB-Apt3 particles was still

remained at 4 8C compared to that at 37 8C (p< 0.05;

Figure 3A). By contrast, after being incubated with the free

aptamermolecules, the uptake efficiencywas significantly

reduced to 15% of that at 37 8C (p< 0.05; Figure 3A). It is

worth mentioning that since the internalization process

was largely blocked by low temperature, the fluorescence

signal was mainly contributed by the particles binding on

thecell surface.These results confirmedthe ligand–receptor

interaction nature between the PB-Apt3 particles andQGY-

7703cells,whichprovidedadriving force for theparticles to

adsorb on cell surface, which was the first and mandatory

step for subsequent uptake.

The possible endocytic pathways for internalization of

exogenous particles were determined by addition of

specific inhibitors.[57,58] The macropinocytosis could be

blocked by amiloride-HCl due to the disturbing of Naþ/Hþ-channels,[59] the clathrin-mediated endocytosis could be

inhibited by amantadine–HCl by preventing budding of

clathrin-coated pits;[60] the caveolae-mediated uptake

would be blocked by genistein through the blocking of

13, 13, 1413–1421


0

10

20

30

40

50llec rep ytisnetni e cne cs eroulf egarevA

Control PB PB-Apt3@QGY PB-Apt3+Apt

***

(A)

0 20 40 60 80 100 120 140

PB-Apt3@Hepli

**

Average fluorescence intensity per cell (%)

PB@Hepli***

*

**

**

Control

AmanAmilo

GeCytD

Control

AmanAmilo

GeCytD

0 20 40 60 80 100 120 140

*

PB-Apt3@QGY

**

Average fluorescence intensity per cell (%)

PB@QGY**

**

**

**

Control

AmanAmilo

GeCytD

Control

AmanAmilo

GeCytD

(C)(B)

Figure 3. A) Influence of free aptamer on the uptake of PB and PB-Apt3 particles by QGY-7703 cells at 4 8C. Influence of pharmacologicalinhibitors on the uptake of PB and PB-Apt3 particles by B) Hepli and C) QGY-7703 cells. The cells were culturedwithout or with pretreatmentby amantadine–HCl (Aman, 1� 10�3 MM, inhibitor of clathrin-mediated endocytosis), genistein (Ge, 100� 10�3 M, inhibitor of caveolae-mediated endocytosis), amiloride–HCl (Amilo, 2� 10�3 M, inhibitor of macropinocytosis), and cytochalasin D (CytD, 10mgmL�1, inhibitor ofcytoskeleton) for 1 h before the particleswere added, respectively. Asterisk indicates significant difference at p<0.05 level versus respectiveinhibitors-free control.

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1418

Src-family tyrosine kinases,[61] and the cytoskeleton could

be destroyed by CytD, which had a strong impact on

particles transportation in cells. Figure 3B shows that the

cell uptake by the Hepli cells was obviously blocked by all

the inhibitors, suggesting that the macropinocytosis,

clathrin-mediated endocytosis, and caveolae-mediated

endocytosis were responsible for the ingestion of both

types of PB and PB-Apt3 particles. The amiloride and

amantadine exhibited a significantly stronger inhibition

effect on the uptake of PB-Apt3 particles than PB particles,

suggesting that the macropinocytosis and clathrin-medi-

ated endocytosis played a more important role in the

internalizationof thePB-Apt3particlesby theHepli cells. As

shown in Figure 3C, the cellular uptake of both types of the

PLGAparticles by theQGY-7703 cellswas largelyblockedby

all the reagents except of genistein, suggesting that the

internalization of the PLGA particles was mediated by the

multiple pathways including macropinocytosis and cla-

thrin-mediated endocytosis rather than the caveolae-

mediated pathway. However, amantadine, amiloride,

and CytD exhibited a significantly stronger inhibition

effect on the cell uptake of the PB-Apt3particles than the PB



particles, suggesting that clathrin-mediated endocytosis,

macropinocytosis, and cytoskeleton played a more impor-

tant role in the internalization of the PB-Apt3 particles by

the QGY-7703 cells.[62] In summary, both the PB and the PB-

Apt3particleswere ingestedby theQGY-7703and theHepli

cells through multiple pathways. Moreover, the clathrin-

mediated endocytosis (receptor–ligand interaction) and

macropinocytosis contributed significantly to theuptakeof

aptamer-decorated particles in both types of cells, proving

that the surface-tethered aptamer molecules were able to

alter the cellular uptake pathways to some extent.

3.4. Intracellular Distribution

Different uptake pathwaymay result in distinct intracellu-

lar distribution of the particles. Usually, the particles

ingested via clathrin-mediated pathway and macropino-

cytosis initially reside in endosomes andmacropinosomes,

respectively, which are acidified and fused with lysosomes

afterwards. The particles ingested via caveolae-mediated

endocytosis will be incorporated into caveolar vesicles

which can be delivered to caveosomes, avoiding

3, 13, 1413–1421


Figure 4. A,B) CLSM images of Hepli cells and C,D) QGY-7703 cells. incubated with50mgmL�1 PB particles (A, C) and PB-Apt3 particles (B, D) for 4 h, respectively. Column 1:merged fluorescence images from Column 2–4, Column 2: endosomes (green), Column3: PLGA particles (red), and Column 4: cell nucleus (blue). Scale bar 10mm. Arrowindicates the overlap of endosome and particles.


www.mbs-journal.de

entrapment in lysosomes.[62] Since the

surface chemistry of the particles can

influence the cellular uptakepathways, it

is reasonable to suspect it also can

influence the particle intracellular traf-

ficking. It is known that ligandmolecules

usually interact with membrane recep-

tors, participate in membrane bound

vesicles, and travel through cells in

different manners to allow the accumu-

lation in early endosomal fractions or

lysosomally associated fractions.[56]

Therefore, the internalization process

and the cellular distribution of the PLGA

particlesweremicroscopicallymonitored

by staining the early endosomes with

anti-EEA-1, the lysosomal compartments

with LysoTracker Green, and the cell

nuclei with DAPI, respectively. Our previ-

ous study in a transwell model has ruled

out the potential release of dye from NR-

loaded particles, and thus the red fluores-

cent signals surely represented the NR-

loaded particles.[48]

Firstly, the distribution of the particles

and endosomes inside cells was observed

at 4h because the endosomeswould fuse

with lysosomes at a later stage.[62–64]

Images of 1mm thick sections in the

middle of cells were obtained by CLSM to

ensure the signals came from intracellu-

lar region. As shown in Figure 4, the

majority of the PB and PB-Apt3 particles

were located inside the QGY-7703 and

Hepli cells rather than adsorbed onto the cell membranes,

confirming the internalization of the particles. The yellow

color from the merged images indicates the overlap of

particles and endosomes (arrow indicated), suggesting that

a part of the internalized particles were resided inside

endosomes, whichwas consistentwith the involvement of

the clathrin-mediated endocytosis pathway.[62] However,

there was no obvious difference between the distributions

of PB and PB-Apt3 particles inside both types of cells at this

stage.

After 8 h incubation, more particles were located inside

the cells (Figure 5). For both cells, a small fraction of

the particles were overlapped with lysosomes at this stage

(yellow color, arrow indicated). This implies again that the

clathrin-mediated endocytosis and the macropinocytosis

participated in the particle uptake for both cells. Mean-

while, most of the internalized PB and PB-Apt3 particles

were well-dispersed in the cytoplasm. This fact would

suggest that either the escapeof themost particles fromthe

lysosomes or the possibility that particles did not enter into



the lysosomes at this stage. Besides, the PB-Apt3 particles

tended to accumulate around the cell nuclei in the QGY-

7703 cells after 8 h co-incubation (Supporting Information,

Figure S5). Although many PB-Apt3 particles were found

around the nuclei, very few colocalization signals of the

particles and cell nuclei could be recorded, suggesting that

the particles could not penetrate through the nucleus

membrane.

4. Conclusion

Using the specific targeting feature of aptamer AS1411 to

thenucleolinand theanti-proteinadsorptionnatureofBSA,

the aptamer-modified PLGA (PB-Apt) particles were pre-

pared and their selectivity of cellular uptake and intracel-

lular transportation were studied. For this context, the

aptamer AS1411 molecules were covalently conjugated

onto PLGA–BSA particles with a diameter of �400nm

in medium, resulting in the aptamer density of 1.9

13, 13, 1413–1421


Figure 5. A,B) CLSM images of Hepli cells and C,D) QGY-7703 cells incubated with50mgmL�1 A,C) PB particles and B,D) PB-Apt3 particles for 8 h, respectively. Column 1:merged fluorescence images from column 2,3, column 2: lysosomes (green), and column3: PLGA particles (red). Scale bar 10mm. Arrow indicates the overlap of lysosome andparticles.

www.mbs-journal.de


1420

(1 molecule/10 nm2) and 3.3mg (1.7 molecule/10 nm2)

per mg PLGA particles depending on the feeding concen-

tration of aptamer. The PB-Apt nanoparticles were prefera-

bly ingested with larger amount and faster rate by liver

cancer cells that over express the nucleolin, the targeting

motif for the aptamer AS1411. Although the PLGA particles

were internalized through multiple pathways, the macro-

pinocytosis and clathrin-mediated endocytosis played a

more important role in the uptake of the PB-Apt particles.

The clarified mechanisms were beneficial for insight

understanding of the interactions between targeting

Macromol. Biosci. 2013, 13, 1413–1421

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhe

particles and cells/cell organelles, and

were expected to extend to other similar

systems.

Acknowledgments: This studywas supportedby the Natural Science Foundation of China(Nos. 51120135001 and 51003094), the Na-tional Basic Research Program of China(2011CB606203), PhD Programs Foundationof Ministry of Education of China(20110101130005 and 20100101120034),and ‘‘Qianjiang’’ outstanding researcher fund-ing of Zhejiang Province (J20110541).

Received: March 28, 2013; Revised: June 9,2013; Published online: July 19, 2013; DOI:10.1002/mabi.201300165

Keywords: aptamer; cellular uptake; particles;pathways; poly(D,L-lactide-co-glycolide)

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study of the selective uptake progress of aptamer-modified plga particles by liver cells

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