Influence of surface coatings of poly(d,l-lactide-co-glycolide) particles on HepG2 cellbehavior and particle fateDahai Yu, Yuying Zhang, Guangyang Zou, Xiaojing Cui, Zhengwei Mao, and Changyou Gao

Citation: Biointerphases 9, 031015 (2014); doi: 10.1116/1.4894531 View online: http://dx.doi.org/10.1116/1.4894531 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/9/3?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) and poly(ethylene glycol)surface films: An in vitro cell culture study Biointerphases 9, 031003 (2014); 10.1116/1.4878461 Arginine-glycine-glutamine and serine-isoleucine-lysine-valine-alanine-valine modified poly(l-lactide) films:Bioactive molecules used for surface grafting to guide cellular contractile phenotype Biointerphases 9, 029002 (2014); 10.1116/1.4864432 Stochastic dynamics of small ensembles of non-processive molecular motors: The parallel cluster model J. Chem. Phys. 139, 175104 (2013); 10.1063/1.4827497 Surface modification of poly(l-lactic acid) with biomolecules to promote endothelialization Biointerphases 5, FA32 (2010); 10.1116/1.3467508 Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, andfirst applications Biointerphases 1, 156 (2006); 10.1116/1.2431704

Influence of surface coatings of poly(D,L-lactide-co-glycolide) particleson HepG2 cell behavior and particle fate

Dahai Yu, Yuying Zhang, Guangyang Zou, Xiaojing Cui, and Zhengwei Maoa)

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Scienceand Engineering, Zhejiang University, Hangzhou 310027, China

Changyou Gaoa)

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Scienceand Engineering, Zhejiang University, Hangzhou 310027, China and State Key Laboratory of Diagnosisand Treatment for Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University,Hangzhou 310003, China

(Received 22 May 2014; accepted 22 August 2014; published 5 September 2014)

This study is focused on the intracellular fate of poly(D,L-lactide-co-glycolide) (PLGA) particles

with different surface coatings after cellular uptake, and their influence on the functions of human

liver cancer cells (HepG2 cells). The PLGA particles coated with polyethyleneimine (PEI) and

bovine serum albumin (BSA) with a similar diameter of �400 nm but different surface chemistry

were prepared. The intracellular distribution of the PLGA particles was also largely dependent on

their surface coatings. The PLGA-PEI particles were removed from cells by exocytosis with a

slower rate compared to the PLGA-BSA particles. In general, uptake of both types of the PLGA

particles did not cause apparent impedance on cell viability and cell cycle, but uptake of the

PLGA-PEI particles did have certain influence on cell functions such as intracellular level of reac-

tive oxygen species, cytoskeleton organization, cell migration, and secretion levels of triglyceride.VC 2014 American Vacuum Society. [http://dx.doi.org/10.1116/1.4894531]

I. INTRODUCTION

Poly(D,L-lactide-co-glycolide) (PLGA) nano/micropar-

ticles are potential delivery vehicles for many drugs such as

antibiotics, chemotherapeutic agents, peptides, proteins,

genes, and vaccines.1–4 In order to optimize their biological

performance, a lot of studies have been focused on the sur-

face modification of PLGA particles with cationic polymers

or ligand molecules to enhance their accumulation in cells

and targeted organs.5–7

It is reasonable to suspect that the surface characteristics

such as chemistry, charge, and the presence of ligand mole-

cules will alter their interaction with cells. On one hand, the

endocytosis process including kinetics and pathways is

known highly dependent on the surface characteristics of the

particles.8 For example, the attachment of folic acid or

aptamer molecules onto PLGA particles helps to increase

uptake in cancerous cell lines and change the uptake path-

ways.9–11 Although a lot of studies have been conducted to

address the impact of surface characteristics of particles on

their cellular uptake, their impacts on particles’ intracellular

fate such as intracellular location and exocytosis are not fully

understood. On the other hand, the cell functions will also be

influenced as a consequence of particle internalization in a

surface characteristic-dependent manner. For example, some

cationic polymers such as polyethylenimine (PEI) and poly-

lysine capped PLGA particles have shown certain cytotoxic-

ity to HEK293 and NIH3T3 cells.12,13 Chang et al. found

significant cytotoxicity of Tween-20 capped PLGA particles

(negatively charged) to brain capillary endothelial cells.14

However, the majority of the studies have been focused on

cell viability rather than other less direct but important func-

tions. Therefore, it is of paramount importance to conduct

case-by-case study on cell-particle interaction and subse-

quent impact on cell functions.

Liver is a vital organ with many functions, including gly-

cogen storage, decomposition of red blood cells, plasma pro-

tein synthesis, hormone production, and detoxification. More

importantly, the intravenous applied particles have to go

through the liver and a large portion of the particles will

accumulate. Therefore, attention should be paid to the inter-

nalization process of colloids into liver-derived cells and its

influence on cell functions. Of these cell lines, the HepG2

cell line, originally isolated by Aden et al. in 1972 from a

primary hepatoblastoma of an 11-year-old Argentine boy, is

the most versatile one. This cell line retains many of the spe-

cialized functions normally lost by primary hepatocytes in

culture such as secretion of the major plasma proteins.15 Hu

et al. found the size, surface charge and configuration of the

poly(ethylene glycol) modified polycaprolactone nanopar-

ticles loaded with paclitaxel have a great effect on the cellu-

lar uptake efficiency and in vitro cytotoxicity to the HepG2

cells.16 Shukla et al. demonstrated that TiO2 nanoparticles

induce significant oxidative DNA damage and cause apopto-

sis to HepG2 cells even at very low concentrations.17

However, a correlation study on the fate of the PLGA par-

ticles with different surface coatings inside HepG2 cells, and

subsequent influence on cell functions has not been con-

ducted so far. In this paper, attention shall be paid to the sub-

cellular distribution and the exocytosis process of PLGA

particles with different surface coatings in HepG2 cells.

a)Authors to whom correspondence should be addressed; electronic

addresses: zwmao@zju.edu.cn; cygao@mail.hz.zj.cn

031015-1 Biointerphases 9(3), September 2014 1934-8630/2014/9(3)/031015/9/$30.00 VC 2014 American Vacuum Society 031015-1

Their impact on cell functions is also studied in terms of cell

viability, intracellular reactive oxygen species (ROS), cyto-

skeleton, cell cycle, cell migration, and secretion of total

cholesterol (TC) and triglyceride (TG). These results will

unveil the interaction between cells and colloidal particles

from cellular and molecular levels, and highlight the impor-

tance of surface coating on particles for intracellular drug

delivery and safety issue.

II. EXPERIMENTAL SECTION

A. Materials

PLGA (LA:GA¼ 75:25, MW¼ 130 kDa), branched poly-

ethylenimine (PEI, MW¼ 25 kDa), bovine serum albumin

(BSA), 20,70-dichlorodihydro-fluorescein diacetate (DCFH-

DA) and 4,6-diamidino-2-phenylindole (DAPI) were pur-

chased from Sigma-Aldrich. Early endosome anti EEA-1,

Lyso TrackerVR Green and ER TrackerVR Green were

obtained from Invitrogen Co., Ltd. Enzyme-linked immuno-

sorbent assay (ELISA) kits for total cholesterol (TC, Product

No. ABIN771240) and triglyceride (TG, Product No.

ABIN577625) detection were obtained from Antibodies-

online.com (Shanghai Xiangsheng Co., Ltd., China as local

distributor). All the chemicals were used as received. Milli-

Q water was used throughout the experiments.

B. Particles preparation

The PLGA particles were prepared by an O/W emulsion-

solvent evaporation method reported previously.8 Briefly, 1

ml 2% (w/v) PLGA dichloromethane solution (organic phase)

was added into 4 ml 5% PEI or 3% BSA solution (water

phase), and then emulsified with an ultrasonicator (MISONIX

Ultrasonic liquid Processors) for 20 s. The obtained emulsion

was poured into 150 ml water, and stirred at room tempera-

ture for 3 h with a magnetic stirrer until the organic solvent

was completely evaporated. The PLGA particles were col-

lected by centrifugation at 12500 rpm for 15 min, and washed

with water 5 times to remove free PEI or BSA in the water

phase. The PLGA particles containing Nile red were similarly

prepared by adding 0.2 mg/ml Nile red into the PLGA solu-

tion before mixing with the PEI or BSA solution. The fluores-

cence intensity of NR-labeled PLGA-PEI and PLGA-BSA

particles was normalized with each other in further quantita-

tive comparison of the cellular uptake study. The concentra-

tion of the particles was determined by weighing the

completely dried particles from 1 ml suspension.

C. Particles characterization

The morphology of the PLGA particles was analyzed by

scanning electron microscopy (SEM, HITACHI S-520) and

transmission electron microscopy (TEM, Philips TECNAL-

10). A drop of the PLGA particles suspension was added

onto a clean glass and copper grid with carbon membrane

for SEM and TEM observations, respectively.

The size and surface charge of the PLGA particles were

determined using Beckman Delsa Nano (Beckman Coulter).

To study the colloidal stability in cell culture medium, the

PLGA particles were incubated in DMEM (Gibco, USA)/

10% (v/v) fetal bovine serum (FBS, Sijiqing Co., Ltd.,

Hangzhou, China) medium for 24 h.

D. In vitro experiments

1. Cell culture

The HepG2 cells were obtained from American Type

Culture Collection (HB-8065, ATCC, USA) and maintained

in regular growth medium consisting of high-glucose

DMEM supplemented with 10% fetal bovine serum, 100 U/

ml penicillin, and 100 lg/ml streptomycin, and cultured at

37 �C in a 5% CO2 humidified environment. The cells used

in this study were passage 15–25 after receiving.

2. Intracellular distribution

The cells were seeded on a 24-well plate at a density of

1� 105 cells per well and allowed to attach for 16 h, which

were then cultured with 50 lg/ml of NR-labeled PLGA par-

ticles (about 9.6� 1011 particles/L) for different time. Early

endosomes, lysosomes, endoplasmic reticulum, and cell

nuclei were fluorescently stained to display the intracellular

distribution of the PLGA particles by confocal laser scan-

ning microscopy (CLSM, TCS SP5, Leica). Briefly, the

HepG2 cells were carefully washed with PBS 3 times, and

then cultured with anti-EEA-1 (for endosome compartment),

Lyso Tracker Green (for lysosomal compartment), ER

Tracker Green (for endoplasmic reticulum) and DAPI (for

cell nucleus) at 37 �C for another 30 min, respectively.

3. Exocytosis of PLGA particles

The cells were seeded on a 24-well plate at a density of

1� 105 cells per well and allowed to attach for 16 h, which

were then cultured with various concentrations of NR-

labeled PLGA particles for 24 h, or cultured with 50 lg/ml

NR-labeled PLGA particles for different time. The cells

were always kept in dark to avoid the bleaching of fluores-

cence. The fluorescent signal of the cells (intracellular parti-

cle amount) was quantified by flow cytometry at determined

time and set as the starting point of the exocytosis. The cells

were then incubated with particle-free culture medium. To

study the influence of the serum and temperature on the exo-

cytosis, the cells were incubated in serum-free medium at

37 �C or in normal medium at 4 �C for 6 h, respectively.

4. Cell viability

The HepG2 cells were plated at a density of 2� 104 cells

per well in a 96-well plate and cultured for 16 h. The me-

dium was replaced with fresh one containing the PLGA par-

ticles of different concentrations. To determine the cell

viability, after co-incubation with the particles for 24 h, 20

ll 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-

mide (MTT, 5 mg/ml) was added to each well and the cells

were further cultured at 37 �C for 4 h. The dark blue forma-

zan crystals generated by the mitochondria dehydrogenase in

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Biointerphases, Vol. 9, No. 3, September 2014

viable cells were dissolved in dimethyl sulfoxide (DMSO),

whose absorbance was measured at 570 nm by a microplate

reader (Biorad Model 680).

5. Reactive oxygen species

The oxidation-sensitive probe DCFH-DA was employed

for determination of the generation of ROS. DCFH-DA is an

amphiphilic nonfluorescent molecule that readily crosses

cell membranes, deacetylated by esterases, and then oxidized

to highly fluorescent 20,70-dichlorfluorescein (DCF) in the

presence of intracellular ROS. In this study, the cells were

seeded in 24-well plates at a density of 1� 105 cells per

well and cultured for 16 h, and then were incubated with

50 lg/ml PLGA particles. After a certain period of time, the

cells were incubated with a complete medium containing 10

lM of DCFH-DA for 30 min at 37 �C in dark. Then the cells

were washed with PBS, trypsinized, collected and analyzed

by flow cytometry. Since the ROS level in normal cells is

quite stable, here we just use normal cells without particles

as control.

6. Secretion of total cholesterol and triglyceride

The cells were seeded on a 24-well plate at a density of

1� 105 cells per well and cultured for 16 h. After being

treated with 10, 50, and 150 lg/ml PLGA particles for 24 h,

the concentrations of TC and TG in the supernatant were

measured by ELISA assays according to the user’s manual.

The assays employ a monoclonal antibody specific for TC

and TG that has been precoated onto a microplate, respec-

tively. Standards and cell culture supernatants were pipetted

into the wells and the immobilized antibody is bound to any

TC/TG present, respectively. After washing away any

unbound substances, an enzyme-linked polyclonal antibody

specific for TC/TG was added to the wells, respectively.

Following a wash to remove any unbound antibody-enzyme

reagent, a substrate was added to the wells. After a predeter-

mined incubation period the color development is stopped

and the intensity of the color is determined at 450 nm and

the results were obtained by comparison with a standard

curve constructed using dilutions of standard samples pro-

vided in testing kits. Each value was averaged from 3 paral-

lel experiments.

E. Statistical analysis

All values were expressed as mean 6 standard deviation

(SD). Statistically significant value was set as p< 0.05 based

on Student t-test.

III. RESULTS AND DISCUSSION

A. Characterization of PLGA particles

The PLGA particles were prepared by an O/W emulsion-

solvent evaporation method with PEI and BSA as stabilizers

in water phase.8–11,18–20 The average sizes of the PLGA-PEI

and PLGA-BSA particles were found to be �420 nm in

water from DLS and �170 nm in a dry state (determined

from 100 particles on TEM images) (Fig. 1 and Table I).

The larger size of the particles in water is attributed to the

hydrophilic PEI and BSA corona on the particle surfaces

whose dry thickness is about 30–50 nm and contribute to

about 4% of the dry mass of the particles.8 Since only one

FIG. 1. SEM (a) and (b) and TEM (c), (d) images of (a) and (c) PLGA-PEI and (b) and (d) PLGA-BSA particles.

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Biointerphases, Vol. 9, No. 3, September 2014

peak can be observed from DLS result of the particles, the

significant aggregation of the particles is ruled out.8 Owing

to the surface coverage of PEI and BSA molecules, the

PLGA-PEI and PLGA-BSA particles were positively (þ40

mV) and negatively charged (�20 mV) in water, respec-

tively (Table I). Merhi et al. proved the importance of cell

culture conditions on physicochemical characteristics of par-

ticles,21 so we also check particles’ characteristics in me-

dium with FBS. The size and surface charge of the PLGA-

BSA particles were largely retained in cell culture medium

containing 10% FBS. However, the PLGA-PEI particles

aggregated very fast and the surface charge turned to nega-

tive due to protein adsorption.8

B. Intracellular distribution

Hydrophobic dye Nile red (NR) was preloaded during the

particle fabrication to make the PLGA particles detectable

via CLSM. It was demonstrated previously that no detectable

NR was released from the particles after incubation in

RPMI-1640/10%FBS in the presence of cells at least for

24 h at 37 �C due to poor solubility of NR in aqueous me-

dium. Additionally, loading of the NR did not bring signifi-

cant influence on particle size and morphology as well as

surface charge and colloidal stability.8

In the cellular endocytosis process, the exogenous par-

ticles are enclosed into endosomes initially, which are

matured into late endosomes or multivesicular bodies and

eventually fused with lysosomes accompanying with a sig-

nificant change of pH.22 Since the endosomes would fuse

with lysosomes at a later stage, we chose 1, 3, and 6 h to

observe the distribution of the particles and endosomes

inside cells. Figure 2 shows that the internalized particles

increased along with the incubation time, most of which

resided inside endosomes judging from the yellow color in

the merged images (column 3 in Fig. 2). The lysosomes and

PLGA particles were also followed after 3 and 12 h incuba-

tion. Figure 3 shows that only a few particles colocalized

TABLE I. Size and surface charge properties of PLGA-PEI and PLGA-BSA

particles.

Sample

Size (nm) Zeta potential (mV)

Water DMEM/10%FBS Water DMEM/10%FBS

PLGA-PEI

particles

420 6 28 1001 6 118 44 6 2 �13 6 0.6

PLGA-BSA

particles

424 6 4 434 6 20 �17 6 4 �17 6 0.5

FIG. 2. CLSM images of HepG2 cells cultured without (a) or with 50 lg/ml PLGA-PEI particles for 1 h (b), 3 h (c), and 6 h (d), and PLGA-BSA particles for 1

h (e), 3 h (f), and 6 h (g), respectively. Column 1: merged fluorescence images showing early endosomes (green) and PLGA particles (red, preloaded with Nile

red); column 2: merged fluorescence images showing cell nucleus (blue) and PLGA particles (red); column 3: merged images of bright fields, early endosomes

(green), PLGA particles (red), and cell nuclei (blue). The HepG2 cells were labeled with early endosome probe (green) and DAPI (blue) for 30 min before

CLSM observation. Scale bar 20 lm.

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Biointerphases, Vol. 9, No. 3, September 2014

with lysosomes after 3 h incubation, since most of the par-

ticles resided inside the endosomes at this moment (Figure

2). After 12 h incubation, a part of the PLGA-PEI particles

were overlapped with lysosomes [yellow color in Fig. 3(b)],

implying that many PLGA-PEI particles did not enter into

lysosomes or had escaped from the lysosomes. By contrast,

quite a few PLGA-BSA particles were trapped inside lyso-

somes [yellow color in Fig. 3(d)], suggesting the surface

property of the particles may dominate their intracellular

transportation. This might be attributed to the different

uptake pathways of the two particles and/or easy escape of

PLGA-PEI particles from lysosomes due to the proton

sponge effect of PEI molecules.23 Quite many PLGA-BSA

particles were overlapped with the endoplasmic reticulum

(yellow color in Fig. 4) after 3 and 12 h incubation.

However, a very few PLGA-PEI particles colocalized with

ER after 12 h incubation. This result indicates again that the

intracellular distribution is influenced by surface chemistry

of the particles. Since continuous ER stress could result in

cell malfunction and even apoptosis,24 attention should be

paid to disclose the impact on cell functions as a result of

particle uptake. Moreover, although many particles were

found around the nuclei, very few colocalization signals of

the particles and cell nuclei could be recorded (Fig. 5) even

after 24 h coincubation, suggesting that the particles cannot

penetrate into the cell nucleus. Taking all the cellular distri-

bution results into consideration, significant difference was

found between the PLGA-PEI and PLGA-BSA particles in

the interaction with lysosomes and endoplasmic reticulum.

FIG. 3. CLSM images of HepG2 cells cultured with 50 lg/ml PLGA-PEI (a)

and (b) and PLGA-BSA particles (c) and (d) for 3 h (a) and (c) and 12 h (b)

and (d), respectively. All pictures are merged fluorescence images showing

lysosomes (green) and PLGA particles (red). Scale bar 10 lm.

FIG. 4. CLSM images of HepG2 cells cultured with 50 lg/ml PLGA-PEI (a)

and (b) and PLGA-BSA particles (c) and (d) for 3 h (a) and (c) and 12 h (b)

and (d), respectively. All pictures are merged fluorescence images showing

endoplasmic reticulum (green) and PLGA particles (red). Scale bar 10 lm.

FIG. 5. CLSM images of HepG2 cells cultured with 50 lg/ml PLGA-PEI (a)–(c) and PLGA-BSA particles (d)–(f) for 3 h (a) and (d), 12 h (b) and (e) and 24 h

(c) and (f), respectively. All pictures are merged fluorescence images showing cell nucleus (blue) and PLGA particles (red). Scale bar 10 lm.

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Biointerphases, Vol. 9, No. 3, September 2014

C. Exocytosis

The fate of particles in cells is of great importance in

understanding the delivery efficiency and toxicity of par-

ticles. Exocytosis is the reverse process of uptake, which is

responsible for the particle removal from the cells. Many

reports have demonstrated that among others the uptake of

particles by cells depends on their size, shape, and surface

properties.8,25,26 However, very little attention has been paid

to the exocytosis of the particles.27,28 The relationship

between exocytosis and physiochemical properties of the

particles has not been carefully studied. In this study the exo-

cytosis of PLGA particles with different surface coatings

was quantified. The fluorescent signals of the cells after they

were pretreated with PLGA particles were followed by FCM

for at most 20 h. During this time frame, the total protein

level, which is linearly correlated to the cell number, did not

change obviously (Fig. S1),29 suggesting the cell duplication

is neglectable. Therefore, the decrease of fluorescent signal

of the cells can only be attributed to the exocytosis of

NR-labeled particles. As shown in Figs. 6(a) and 6(b),

exocytosed fraction of both types of PLGA particles

increased along with the prolongation of culture time after

the cells were treated with 50 lg/ml particles for 24 h. The

exocytosis of PLGA-BSA particles was significantly faster

than that of the PLGA-PEI particles at all time points except

of 1 h (p< 0.05). This might be attributed to the surface

coating of BSA, which plays an important role in exocytosis

process and is able to enhance the exocytosis of nondegrad-

able poly-D-lysine in rabbit alveolar macrophages.30 The

exocytosed PLGA-PEI and PLGA-BSA particles reached to

70% and 85% of the original loading amount after 20 h,

respectively, suggesting that the PLGA-BSA particles are

preferably expelled out by the HepG2 cells. The results also

indicate that most particles will be removed from the cells in

20 h, suggesting the impact on cells induced by particles

would be limited in a shorter period of time.

When the feeding dose of PLGA particles was improved

to 150 lg/ml, the uptake amount of the particles was signifi-

cantly increased (about 3 fold, Fig. S2A). However, the exo-

cytosed fractions were significantly reduced [Fig. 6(c)],

FIG. 6. (a) Average fluorescent intensity of HepG2 cells and (b) Exocytosis percentage of PLGA particles by HepG2 cells as a function of culture time after

the cells were treated with 50 lg/ml PLGA particles for 24 h (which is designated as the time 0 point in these figures). (c) Exocytosis percentage of PLGA par-

ticles (exocytosis time 6 h) after the cells were cultured with 50 lg/ml particles for 24 h (Standard), 3 h (Feeding 3 h) and 150 lg/ml particles for 24 h

(Feeding 150 lg/ml) particles for 24 h, respectively. (d) Exocytosis percentage of PLGA particles (exocytosis time 6 h) after the cells were cultured with 50

lg/ml particles at 37 �C for 24 h in 10% FBS medium (Standard), and in FBS free medium (FBS free), and at 4 �C for 24 h in 10% FBS medium (4 �C), respec-

tively. Data were measured by flow cytometry and averaged to each cell. Asterisk indicates significant difference at p< 0.05 level.

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Biointerphases, Vol. 9, No. 3, September 2014

revealing longer retention time of the particles inside cells.

Reduction of the coincubation time of particles and cells

from 24 to 3 h resulted in the decrease of the uptake amount

to about 50% (Fig. S2A), which in turn leaded to a slower

exocytosis of the particles in 6 h [Fig. 6(c)]. These results

indicate that the exocytosis rate is positively correlated with

the initial particle concentration inside cells.

The exocytosis is an energy-dependent process, too.

Therefore, a serum free medium and low temperature were

used to block the exocytosis. The exocytosed fraction of par-

ticles was significantly reduced in the serum free medium

and under 4 �C [Fig. 6(d), p< 0.05], indicating that the par-

ticles were removed from cells by the exocytosis pathway

rather than physical diffusion.

D. Cytotoxicity

Small particles may have significant impact on cells

because of their large surface area per unit mass.31–34 It has

been reported that the cellular uptake of particles can induce

a series of consequences on cells and result in the alternation

of important cell behaviors, such as apoptosis, proliferation,

adhesion, migration, differentiation, and so on.25,35–39 As a

type of widely used polymer particles in biomedical field

especially in drug delivery, PLGA particles are generally

considered to be safe and accredited as a drug carrier by

Food and Drug Administration (FDA, USA). However, the

toxicity of the particles is also largely dependent on their

surface properties.12,13 In this study, the viability of HepG2

cells exposed to particles was assessed by MTT assay. As

shown in Fig. 7, in general, the cell viability was slightly

reduced to about 85% of the particle-free control regardless

of the particle type and feeding concentration, suggesting

that uptake of both types of PLGA particles did not cause

significant impact on cell viability.

Furthermore, the substantial but far-reaching cytotoxicity

such as influence on cell functions can not be completely

reflected by the cell viability. Therefore, influence of cellular

uptake of the PLGA particles on cell mortality, cell cycle, in-

tracellular ROS level, cytoskeleton organization, cell adhe-

sion, and migration were studied. As shown in Table S1 and

Figs. S3 and S4, these results suggest that the cell mortality,

FIG. 7. Relative cell viability (normalized to that of the particle free control)

as a function of particle concentration with a culture time of 24 h,

respectively.

FIG. 8. Representative fluorescence images showing the intracellular ROS of HepG2 cells cultured without (a) or with 50 lg/ml PLGA-PEI (b) and (c) and

PLGA-BSA particles (d) and (e) for 4 h (b) and (d) and 24 h (c) and (e), respectively. Scale bar 200 lm. (f) Average fluorescence intensity quantified by flow

cytometry. DCFH-DA (10 mM) was used to stain cells at 37 �C for 30 min in dark. Asterisk indicates significant difference at p< 0.05 level.

031015-7 Yu et al.: Influence of surface coatings of PLGA particles on HepG2 cell behavior 031015-7

Biointerphases, Vol. 9, No. 3, September 2014

cell cycle, cytoskeleton, and cell adhesion ability were not

significantly influenced after the cells were exposed to both

types of particles. However, uptake of the PLGA particles,

especially the PLGA-PEI particles, did cause some impact

on intracellular ROS level and cell mobility (Figs. 8 and S5).

For example, DCFH-DA is a cell-permeable nonfluorescent

probe which is hydrolyzed by intracellular esterases, which

is then trapped in cell. This nonfluorescent molecule can

then oxidized by ROS to the fluorescent dichlorofluorescin

(DCF). The DCF’s fluorescent intensity is a sensitive indica-

tor of intracellular ROS. As shown in Fig. 8, exposure to 50

lg/ml PLGA-PEI particles for 4 h resulted in enhanced intra-

cellular ROS level (p< 0.05). By contrast, exposure to the

same concentration of PLGA-BSA particles resulted in

decreased ROS level (p< 0.05). The intracellular ROS level

of the cells decreased after cocultured with both types of

PLGA particles for 24 h (p< 0.05).

E. TC and TG syntheses

Liver is a vital organ which plays a major role in metabo-

lism, where the total cholesterol (TC) and triglyceride (TG)

are synthesized and stored.40 The cholesterol not only plays

a significant role in maintaining the structure and function of

cell membranes, but also is a kind of raw material for synthe-

sis of bile acid, vitamin D, and steroid hormones. TG mainly

participates in energy metabolism, and provides energy for

body. Therefore, expression of TC and TG is the important

functions of liver cells, i.e., HepG2 cells, which can be used

as an index to assess the health state. As shown in Fig. 9, in

general, there was no significant difference of TC secretion

level between the particle-free control cells and the cells

pretreated with both types of PLGA particles (p> 0.05).

The HepG2 cells secreted more TG after uptake of the

PLGA-BSA particles compared with those treated with the

PLGA-PEI particles. But this difference is not significant in

statistics (p> 0.05). In conclusion, the majority of the impor-

tant cell functions tested in this study was well preserved af-

ter they were exposed to the PLGA particles, which might

be attributed to the fast exocytosis of the particles (Fig. 6).

IV. CONCLUSION

The PLGA particles with a similar size but different sur-

face coating were used to study their fate inside HepG2 cells

and subsequent impact on the cell functions. Quite a few

PLGA-BSA particles were transferred into the lysosomes

and interacted with endoplasmic reticulum. In contrast, very

few PLGA-PEI particles interacted with lysosomes and

endoplasmic reticulum. Exocytosis of the internalized PLGA

particles took place quickly and was dependent on the intra-

cellular dose of the particles. The PLGA-PEI particles were

removed from cells with a slower rate compared to the

PLGA-BSA particles. Generally speaking, the cell functions

were not significantly influenced by the uptake of both par-

ticles. However, uptake of the PLGA-PEI particles did cause

some impacts on intracellular ROS level and cell mobility,

likely due to their longer retention time in the cells.

ACKNOWLEDGMENTS

Financial supports by the Natural Science Foundation of

China (Nos. 51120135001 and 21374097), the National

Basic Research Program of China (2011CB606203), and

Ph.D. Programs Foundation of Ministry of Education of

China (20110101130005).

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