Folic Acid Modified Poly(lactide-co-glycolide) Nanoparticles, Layer-by-Layer Surface Engineered for Targeted Delivery

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Folic Acid Modified Poly(lactide-co-glycolide)Nanoparticles, Layer-by-Layer SurfaceEngineered for Targeted Deliverya

Jie Zhou, Gabriela Romero, Elena Rojas, Sergio Moya,* Lie Ma,Changyou Gao*

The layer-by-layer (LbL) assembly technique was applied for the surface modification ofbiodegradable poly(lactide-co-glycolide) nanoparticles (NPs), employing poly(acrylic acid)(PAA), and polyethylenimine (PEI) as building blocks. Amino terminated poly(ethylene glycol)(PEG) and folate decorated PEG (PEG-FA) were grafted ontothe multilayers via condensation between carboxylicgroups and amine groups from PEG or PEG-FA. The LbLassembly and the covalent functionalization were mon-itored by means of z-potential measurements and thequartz crystal microbalance with dissipation technique(QCM-D). Protein adsorption after incubation of the NPs inculture medium containing optionally the serum proteinswas investigated and related to cellular uptake. Exper-iments on cellular uptake showed that after PEGylationthe uptake ratio of the NPs decreased significantly, butbecame three times larger when PEG-FA was grafted onthe NPs instead of the PEG.

Introduction

Polymeric nanoparticles (NPs) in the submicro size are

promising carriers for controlled drug delivery. Size,

C. Gao, J. Zhou, L. MaDepartment of Polymer Science and Engineering, ZhejiangUniversity, Hangzhou 310027, ChinaFax: (þ86) 571 87951108; E-mail: [email protected]. Moya, J. Zhou, G. Romero, E. RojasCIC BiomaGUNE, Paseo Miramon 182 Ed. Emp. C, San Sebastian,SpainFax: (þ34) 943005301; E-mail: [email protected]

a : Supporting information for this article is available at the bottomof the article’s abstract page, which can be accessed from thejournal’s homepage at http://www.mcp-journal.de, or from theauthor.

Macromol. Chem. Phys. 2010, 211, 404–411

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

amount of loaded material, and release features can be

well controlled for these NPs. Besides that, polymeric NPs

can be further endowed to target specific organs and

tissues, and in many cases are capable of overcoming

certain biological barriers such as the blood–brain

barrier.[1–3] The lactic acid (LA) homopolymer (PLA)[4,5]

and its glycolic acid (GA) copolymer [poly(D,L-lactide-co-

glycolide) (PLGA)][6–9] are among the most frequently used

polymers for the templationofdrugcarriersbecauseof their

good biocompatibility, biodegradability and because the

rate of degradation and drug release from the PLGANPs can

be easily manipulated adjusting the ratio of LA to GA.[6]

In the design of a drug carrier for controlled release, there

are two main aspects, which should be considered. First,

carriersmustbedesigned in suchaway that the release rate

of the encapsulated drugs can be controlled. This can be

generally achieved by adjusting the polymer structures,

DOI: 10.1002/macp.200900514

Folic Acid Modified Poly(lactide-co-glycolide) Nanoparticles . . .

thepreparation conditions or by choosing carrier capable to

release content in certain environments, i.e., due to

hydrolysis at a certain pH or due to a change in the

structure with the temperature. In the case of PLGA

particles, e.g., the rate of drug release can be largely decided

by the preparation method,[10] particle size,[11,12] LA/GA

ratio,[6] release media,[13] and temperature.[14]

The second important aspect is the control of the release

site, generally recognized as the targeting site in order to

enhance the pharmaceutical efficiencywhile side effects are

reduced. The targeted delivery of the drug carriers is usually

realized by attaching specific recognition functions to the

particles, e.g., specificmonoclonal antibodies (mAB)[15–19] or

a specific ligands to certain cell membrane receptors.[9,20–22]

To minimize the unspecific interactions, molecules with

anti-fouling properties such as poly(ethylene glycol) (PEG)

are often used as a spacer between the particle surface and

the recognition function.[19,21,22] For example, McCarron

et al.[16] prepared camptothecin loaded PLGA NPs with

conjugated anti-Fas human mAB and targeted to colon

cancer cells-HCT116. The anti-Fas modified NPs showed a

higher uptake ratio than the blank control. After 90min

incubation, more than 90% cancer cells were killed with the

anti-Fas human mAB conjugated NPs after 48h incubation.

Folic acid (FA) shows extremely high affinity to the folate

receptors (FRs). Comparewith antibodies,molecules like FA

are more stable against harsh preparation conditions, and

thereby are easier to handle andmore convenient formany

applications. FA is a kind of B vitamin necessary for the

production andmaintenance of new cells. It is well known

that a low level of FRs on the cellmembrane is expressed by

normal tissues, but the FRs are overexpressed by many

human tumors. Therefore, the FAgraftedNPs canbeused to

specifically target tumor tissues. Actually, NPs graftedwith

FA have been used for imaging[23] and killing tumor

tissues.[20,21,24] ZhaoandYung[21] graftedFAontoPLGA-PEG

copolymers, which could be assembled into micelles with

the anticancer drug-doxorubicin (DOX). Loaded PLGA

micelles modified with FA were more effective to kill

cancer cells thancontrolmicelleswithoutFAbut showedno

significant increase in cytotoxicity for normal human

fibroblasts. Also, Muregesan et al. prepared folate con-

jugated PEGylated PLGA NPs as carriers for the anticancer

drug Docetaxel. They show two different ways of conjuga-

tionof theFAto thesurfaceof theNPswithdifferentdensity

of folic groups and evaluated cytotoxicity for the carriers.

The folate groups were either attached covalently to PLGA-

PEG block copolymers or covalently bounded to the NPs

after particle preparation.[25]

In this work, we have prepared PLGA NPs by an O/W

emulsion–solvent evaporation method with polyethylenei-

mine (PEI) in thewaterphaseas stabilizer. The layer-by-layer

(LbL) assembly,[25,26] which is well know technique for

surfacemodification,wasused to engineer the surface of the



NPs. Then, the multilayer coated PLGA NPs were further

modifiedwithFAattachedtoaPEGspacer.TheLbLtechnique

can sequentially modify the surface of the NPs without

affecting their stability. The assembled multilayers provide

chemical groups, to which functional molecules can be

covalently immobilized.AstepwisemodificationofthePLGA

NPs and the characterization of their properties along with

the assembly have been carried out. The assembly and

covalent binding of PEG and PEG-FA process has been

followed by z-potential measurements. Protein adsorption,

which is related to cellular uptakehas beenmeasured by the

BCAassay, and z-potentialmeasurements. Cellularuptakeof

the PLGANPs has been characterized byflow cytometry and

confocal laser scanning microscopy (CLSM).

Experimental Part

Materials

Poly(D,L-lactide-co-glycolide) (PLGA) (D,L-lactide 90: glycolide 10),

average molecular weight of 100 kDa, was purchased from the

China Textile Academy. Poly(acrylic acid) (PAA, Mw�10kDa),

branched PEI (Mw� 25kDa), FA, Jeffamine ED-2001 (amino

terminated PEG, Mn�1.9 kDa), phosphate buffer saline (PBS),

ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide

(NHS), PIERCE BCA Protein Assay Kit, Dulbecco’s Modified Eagle’s

Medium (DMEM), fetal bovine serum (FBS) were purchased from

Sigma–Aldrich. The cell line HepG2 was purchased from the

American Type Culture Collection (ATCC). All chemicals were used

as received.

Synthesis of Folic Acid (FA) Grafted Poly(ethylene

glycol) (PEG)

First, 65mg (0.15mmol) FAwas dissolved in 2.5mL dimethylsulf-

oxide (DMSO) overnight. Then, 38mgNHS (0.33mmol) and 30mg

EDC (0.17 mmol) were added into the solution to activate COOH

groups of FA. The final molar ratio of FA/NHS/EDC was 1:2.2:1.1.

The reaction was left for 18 h and afterwards 300mg of amino

group terminated PEG (0.15 mmol) were added to the solution.

After 24 h, the product of reaction was dialyzed against water for

1week in a dialysis bagwith a cut offMw of 1 000, replacingwater

every 24 h. The final product was lyophilized with a lyophilizer

(Christ alpha 1-2 LD plus, Germany). The FA grafted PEG (PEG-FA)

was characterized by FTIR (Nicolet 6700 FT-IR, UK) and 1H NMR

(500 M, in D2O).

Preparation of Poly(D,L-lactide-co-glycolide) (PLGA)Nanoparticles (NPs)

Poly(D,L-lactide-co-glycolide) (PLGA)NPswerepreparedbymeansof

an O/W emulsion–solvent evaporation method.[8] Firstly, 1mL of

20mg �mL�1 PLGA dichloromethane solution (organic phase) was

www.mcp-journal.de 405

J. Zhou, G. Romero, E. Rojas, S. Moya, L. Ma, C. Gao

406

added to 4mL 5% PEI solution (water phase) and then emulsified

with an ultrasonicator (Sonics VCX500) for 20 s. This emulsionwas

poured into 100mL distilled water, and stirred for 3 h with a

magnetic stirrer until the organic solvent was totally evaporated.

The PLGA NPs were collected by centrifugation at 10 000� g for

5min, andwashedwithmilliQwater five times to remove the free

PEI initially presented in the water phase. PLGA NPs containing

rhodamine 6G (Rd6G) were similarly prepared by addition of

0.5mg �mL�1 Rd6G into the PLGA solution before mixing with the

PEI solution.

Layer-by-Layer (LbL) Assembly and Folic Acid (FA)

Immobilizing on Nanoparticles (NPs)

1mg �mL�1 PAA and PEI solutions in 0.5M NaCl were employed for

the LbL coating. The pH of the solution was adjusted to 7.4 by

addition of either 1M HCl or NaOH. For the LbL coating of the PLGA

NPs, the incubation time of each polyelectrolyte layer was 15min,

and the NPs were washed with a 0.5M NaCl solution three times

between each layer. After five layers of PAA and PEI (PAA as the

outmost layer) were deposited, the multilayers were either only

crosslinked or crosslinked and PEG grafted following the method

described by Meng et al.[27] The NPs with multilayers were

suspended in 10� 10�3M EDC and 10�10�3

M NHS solution

(pH 5.6) 30min to activate carboxylic groups, and then in a

10� 10�3M EDC and 10� 10�3

M NHS (pH 8.6) or 10� 10�3M EDC,

10� 10�3M NHS and 5mg �mL�1 of amine terminated PEG or PEG-

FA solution (pH 8.6) for crosslinking, PEGylation or PEG-FA

immobilization, respectively. 40h later, the NPs were rinsed with

MilliQ water.

Characterization of the Layer-by-Layer (LbL)

Multilayer Assembly and Chemical Modification

The z-potential of the PLGA NPs either coated with PEI/PAA

multilayers or after crosslinking were measured in 10mM NaCl

(pH¼7.4) with 60V voltage with a Zetasizer nano Malvern.

An E4 Quartz Crystal Microbalance with Dissipation (QCM-D)

fromQ-Sense,Goteborg, Swedenwas employed to followmass and

dissipation changes during the crosslinking and PEGylation

process. Gold coated quartz crystals (5MHz) were used as

substrates. (PEI/PAA)3 layerswere assembled on thequartz crystals

following the process mentioned above.

Protein Adsorption on the Layer-by-Layer (LbL) Coated

Nanoparticles (NP)

Todetermine theamountofproteinsadsorbedontheNPs,3mgNPs

were accurately weighted and incubated in 1mg �mL�1 Bovine

Serum Albumine (BSA) in PBS for 2 h or in DMEM culture media

with 10% FBS for 2 and 16h at 37 8C, respectively. The supernatantwas discarded after centrifugation and the particles were washed

three times with 10�10�3M PBS. 1mL 5% SodiumDodecyl Sulfate

(SDS) was then added. The system was sonicated 40min at 40 8C.The amount of adsorbed proteins was measured from the

supernatant by the BCA protein assay kit.



The z-potential of the NPs after protein adsorption was

measured as described above.

Cellular Uptake

TheHepG2 cell line, a kindof human liver cell lines,was cultured in

DMEMwith10%FBSand1000Upenicillin, 10mg/ml streptomycin

at37 8Cand in5%CO2.Whenthecell confluencewasaround70%,all

thecellsweretrypsinized.300 000cellswereseed intoeachwellofa

24-well plate. 24h later, PLGANPswith Rd6G and different surface

coatings were added into the culture medium. The final

concentration was adjusted to 100mg �mL�1. Following different

incubation times: 0.5, 1, 2, 4, 8, 12, and 24h, the cells were washed

with PBS twice, trypsinized and studied with flow cytometry (BD

FACSCanto II). Flow cytometry data were analyzed with the

WinMDI2.9 program.

Confocal laser scanning microscopy (CLSM, Carl-Zeiss LSM 10

META) was used to image the cells after incubation with NPs.

100 000 cells were seeded on a coverslip which was placed in a

3.5 cm culture plate. 24 h later, PLGA NPs with different surface

coatingsandRd6Gwereadded into theculturemediumwithafinal

concentrationup to 50mg �mL�1. After incubation for another 12h,

the culture medium was removed and the cells were rinsed three

times with sterilized PBS. The cells were subsequently fixed with

3.7% formaldehyde solution in PBS for 30min. Finally, the coverslip

was sealed with Mounting Medium for fluorescence with DAPI

(Vector H-1200) and observed under CLSM employing a 63� oil

objective.

Results and Discussion

PLGA NPs were prepared by an O/W emulsion–solvent

evaporation method with PEI in the water phase[28] as a

stabilizer for the dispersion. The average size of the

particles with PEI as stabilizer is around 350nm with a

narrow distribution in the wet state as shown in the

supporting information (Figure S1a of Supporting Informa-

tion) and around 250nm in a dry state (Figure S1b of

Supporting Information). This difference in the measured

size is due to the hydrophilic PEI molecules on the particle

surface.[28] Their surface is positively charged with a

z-potential of þ35mV. PLGA NPs will be modified with

a multilayer of PEI and PAA. The multilayer will

be later crosslinked and PEGylated via amidation of the

amines in the PEG terminates with the �COOH in the PAA

chains.

The z-potential of the NPs was measured to prove

polyelectrolyte assembly and the covalent binding of PEG

and PEG-FA on the PLGA NPs. The alternative assembly of

polyelectrolytes of opposite charge induces surface rechar-

ging and the consequent change in the signof the potential.

During the LbL assembly of PEI/PAA the z-potential

oscillated between þ35� 5mV and �35� 5mV for either

PEI or PAA as outmost layer.[29] After crosslinking or

DOI: 10.1002/macp.200900514


PEGylation, the z-potentials changed to �20mV and

�11mV, respectively (FigureS2ofSupporting Information).

This is understandable since the negatively charged

carboxylic groups were used for the crosslinking. The

further decrease in the value of the potential observed after

PEGylation is brought by the hydrodynamic screening

effect of the PEG molecules.[30] After PEG-FA grafting,

however, the z-potential varied to �22mV again. We

attribute this value to the additional carboxylic groups

brought to the NPs surface by the FA molecules.

To have an additional proof of the covalent binding

taking place on the multilayers we followed the cross-

linking and condensation reactions on planar multilayers

by means of the QCM-D. A multilayer of PEI and PAA was

previously assembled on top of a silica coated QCM-D chip.

The assembly was also followed in the QCM-D device, data

not shown. In Figure 1 we can observe the changes in

frequency and dissipation during crosslinking and PEGyla-

tion. PEI/PAAwere first stabilized in water for 5min. Then,

water was exchanged by the 10� 10�3M EDC and

10� 10�3M NHS solution (pH 5.6) (activation solution).

During activation before crosslinking and PEGylation the

frequency did almost not change. 30min after addition the

activation solution was exchanged by either the cross-

linking solution (CLS) or the PEGylation solution. In both

cases the samples were incubated for 40h. Following

incubation in the CRL, a final decrease in 5Hzwas recorded

for the frequency and a dissipation increase of only 0.4 U.

The crosslinking does not induce a significant change in the

multilayermass. The slight increase inmass could bedue to

an increase in thewater entrapped in the layers as result of

Figure 1. Frequency and dissipation changes during crosslinkingand PEGylation of PEI/PAA multilayers followed by QCM-D.



polyelectrolyte reorganization during crosslinking. After

PEGylation adecrease in 25Hz in frequency andan increase

in 4 U in dissipation was observed. The decrease in

frequency is higher than for crosslinking and can be

associated with a mass increase in the multilayer as result

of the binding of PEG. QCM-D confirms, therefore, that

PEGylation takes place on the multilayer.

For target drug delivery, it is very important to increase

circulation time after the NPs are injected into the blood, in

order to reach their targeted tissues or organs. Normally,

after the injection, there are some proteins in serum

including aolipoprotein E[31] or complement C3[32] are

unspecifically adsorbed on those NPs, which can trigger

macrophage recognition and uptake. By decreasing the

interaction of the NPs with these specific proteins it is

possible to increase the circulation time and reduce

unspecific uptake of NPs.

We detected protein adsorption on the PEI/PAA multi-

layers coated PLGA NPs with the BCA method employing

twodifferentmediums: a1mg �mL�1 BSA in10mMPBSand

amediaofDMEMwith10%FBS. Figure2 shows that theNPs

covered with the (PAA/PEI)2 multilayers had the largest

proteinadsorptionamount,50–70mg �mg�1NPs, regardless

of the composition of the medium. Protein adsorption for

the (PAA/PEI)2 coating was even larger than that of the

control NPs, 40–55mg �mg�1 NPs, which also had PEI on

their surface. Theamountof adsorbedproteinson the (PAA/

PEI)2/PAA coated NPs with crosslinked multilayers PEGy-

lated and PEG-FA covered NPs largely decreased to around

20–30mg �mg�1 NPs. In the DMEM/FBS medium generally

more proteins adsorbed regardless of the structures of the

NPs than in the pure BSA protein medium. On the control

NPs and on the (PAA/PEI)2 coated NPs, the amount of

adsorbed proteins slightly decreased when extending the

incubation time from 2 to 16h. For all the samples, the

longer incubation times resulted in an obviously higher

Figure 2. Protein adsorption amount of the PLGA NPs with differ-ent surface properties at variable conditions.



Figure 3. (A) cellular uptake ratio and (B) mean fluorescenceintensity for PLGA NPs with PEI/PAA coating, crosslinked andPEGylated as a function of the incubation time as calculated fromflowcytometry measurements.

408

amount of adsorbed proteins. In particular, the protein

amount on the (PAA/PEI)2/PAA coated NPs was almost

doubled and finally reached a value of 32mg �mg�1 NPs

after 16h incubation. These results imply that although the

protein components on the NPs surface might be changing

all the time during the incubation,[33–35] the overall

adsorption of the proteins on the positively charged

surfaces is rapidly equilibrated, due to charge attraction.

The protein adsorption on the negatively charged surface is

a process, which requires a relatively longer time than for

the positive charged surfaces, though the adsorption

process can be similar for both kinds of surfaces.

z-Potential measurements further confirmed the protein

adsorption on the NPs (Figure S3 of Supporting Iformation)

shows that after incubation in DMEM, for all the samples

measured the absolute value of the z-potential decreased

compared with their initial values due to the increase in

ionic strength. After incubation in DMEM/FBS, however,

the surface charge changed to negative values, confirming

undoubtedly the adsorption of serum proteins, which are

usually negatively charged. This is consistent with the

previous results, since the most abundant and negatively

charged albumin (�36–50 g � L�1) are adsorbed in less than

5min on the surface.[33] For all the other NPs, the z-

potentials remained negative with very minor changes.

Actually, proteins canadsorbontheNPssurfaceeven if they

are negatively charged or PEGylated, because of the

nonuniform distribution of charges in the proteins and

existence of other forces such as hydrogenbonding and van

der Waals forces. Researchers from Gurny’s group also

found both the bare PLA NPs and PEG-covered particles

adsorbed almost the same amount of albumin from

plasma.[31]

The uptake of the PEI/PAA coatedNPs and of the PEG and

PEG-FA modified NPs by HepG2 cells was investigated by

flow cytometry (Figure S4 of Supporting Information).

Uptake was characterized by two parameters: the cellular

uptake ratio and the fluorescence intensity.

The cellular uptake ratiowas calculated from the dotplot

graph of forward scattering (FCS) versus fluorescence

intensity (PE-A) employing the WinMDI program of data

analysis. Firstly, a threshold of fluorescence was generated

using a control sample, i.e., the HepG2 cells without

exposure to the NPs. All dots corresponding to the control

sample are located at intensities below this threshold

(Figure S4a of Supporting Information). The number of cells

carryingfluorescently labeledNPs is obtained fromthedots

located at higher intensities than the threshold (Figure S4b

of Supporting Information). The cellular uptake ratio equals

to (no. of the dots over the threshold/total no. of the

dots)100%. The fluorescence intensity is defined here as the

mean fluorescence intensity of all the dots in the dotplot,

which reflects the amount of fluorescent NPs associated to

each cell.



Quantitative cellular uptake of the NPs was then

investigated as a function of incubation time. In

Figure 3A the percentage of cells displaying uptake of

NPs has been plotted as a function of the time for the

different surfacemodified NPswhile in Figure 3B themean

fluorescence per cell has been displayed as a function of the

incubation time. Figure 3 shows that both the cellular

uptake rate and thefinal amountofuptakenNPsaregreatly

dependent on the surface chemistry of the NPs. For

example, the uptake rates of the NPs with positively

charged surface [bare NPs and (PAA/PEI)2 coated NPs] were

much faster at the beginningwith an uptake ratio of�40%

within 1h incubation. Then, the uptake rate became slower

and only after incubation for 24hfinally reached anuptake

ratio of the 60%. The fluorescence intensity displayed the

samebehavior (Figure3B). TheNPswithPAAas theoutmost

layer showed a more sustainable increase in both of the

uptake ratio, which increased �20% during the first hour

DOI: 10.1002/macp.200900514


Figure 5. Confocal laser scanning microscopy (CLSM) images ofHepG2 exposed to (A) (PAA/PEI)2, (B) (PAA/PEI)2PAA, (C) PEG,(D) PEG-FA coated NPs for 12 h.

and then 45% in the following 23h, and the fluorescence

intensity, which increased around 100 a.u. during the first

hour and�380 a.u. in the following 23h. Although the final

uptake ratiosof these threeNPswereall around60–65%, the

mean fluorescence intensity of the (PAA/PEI)2/PAA coated

NPs was 50 a.u. higher than the others. On the other hand,

NPs covered with PEGylated multilayers showed the

slowest uptake rate, lowest cellular uptake ratio andmean

fluorescence intensity. Actually, in the first hour no cellular

uptake could be detected, and the final uptake ratio was

only11%with thefluorescence intensityof 40a.u. after 24h

incubation. It was unexpected that after crosslinking, the

cellular uptake of the NPs also significantly decreased to be

less than that of their precursors, i.e., the (PAA/PEI)2/PAA

coatedNPs, andwas only higher than that of the PEGylated

NPs. Immobilization of FA on themultilayers (PEG-FANPs),

however, could significantly enhance the cellular uptake

compared with the PEG NPs. For example, the cellular

uptake ratio after 24h incubation improved to a 45%, i.e.,

3 timesof thePEGylatedNPs. InFigure4wehaveplotted the

mean fluorescence intensity in the fluorescence distribu-

tion against number of cells after 24h incubationof theNPs

with the cells. The highest uptake is shown by the control

and PAA/PEI coated NPs; crosslinked NPs and PEG-FA

modifiedNPs showan intermediateuptake. PEG coatedNPs

show the lowest uptake.

The uptake of PLGA NPs was also studied with Confocal

Microscopy. Micrographs in Figure 5 were taken from the

middle planes of the cells in z direction. PLGA NPs were

labeled with rhodamine 6G as described in the Experi-

mental Part. The cell nucleous was stained with DAPI. The

number of labeled NPs and their distribution in the cell

varied according to the surface composition of the NPs and

were consistent with the flow cytometry measurements

(Figure 3 and 4). Figure 5 shows that the (PAA/PEI)2 coated

NPs (red spots) could beobservedboth in the cytoplasmand

Figure 4. Mean fluorescence intensity per cell after exposed todifferent PLGA NPs for 24 h.



on the cell surroundings (Figure 5A), whereas most of the

(PAA/PEI)2/PAA NPs were internalized into the cytoplasm

(Figure 5B). Very few PEG NPs can be observed in Figure 5C,

where almost no fluorescence coming from rhodamine 6G

canbedetected. In Figure 5Dweobserve that the amount of

PEG-FA NPs increased again. In all the cases, no particles

could be found in the cell nuclei. It must be said that is not

always easy to distinguish betweenNPs attached to the cell

membranes and internalized NPs. It is though clear that

some NPs are closer to the nucleus and can be thus

postulated to be in the cytoplasm.Most likely, there areNPs

both at the surface of the cells and in the cytoplasm. It is

noticeable that negatively charged NPs tend to be found

more in the cytoplasm and that the PEG coated NPs are

present in a much lower amount than when the NPs are

charged. The addition of FA increases the presence of NPs in

the cytoplasm and the cell membrane, providing a

qualitative confirmation of the behavior observed by flow

cytometry.

The cellular uptake is an important cell activity, which is

influenced by many factors such as the chemistry of the

particle surface including charge,[36] ligands,[9,15–18] pro-

teins[31,32,37] etc., particle size,[38] temperature, and also the

particular cell type involved.[38] Considering similar cellular

uptake ratios for the positively charged control and PAA/

PEI2 coated, and negatively charged (PAA/PEI)2PAA coated

NPs (Figure 3), the surface charge does not provide an

appropriate explanation for the observed phenomenon.

Indeed, z-potential measurements reveal that in the cell

culturemedium containing serum, all the particles became

negatively charged regardless of their initial surface

properties, implying protein adsorption occurs neverthe-

less as demonstrated in Figure 2.Moreover, it is known that



410

the surface properties of the NPs will change along with

timebecause the binding strengthbetween theNP surfaces

and the molecules in the medium. The particular proteins

adsorbed play an important role in the cellular

uptake.[31,32,34,35,37] However, the adsorption dynamics,

theadsorbedamountofproteinsandtheparticularproteins

adsorbed must be different on the positive and on the

negative NPs. On the positively charged surface, albumin,

the most abundant protein in serum, adsorbs immediately

to form a comparatively dense layer due to the charge

attraction. Subsequently, other proteins in the serum, e.g.,

apolipoprotein E may further adsorb or replace part of the

albumin after 10min incubation, and then the surface

composition does not change too much after 30min.[33]

The adsorbed protein apolipoprotein E has been proved to

bind to a specific receptor located in the liver.[37] On the

negatively charged NPs such as the (PAA/PEI)2/PAA coated

and crosslinked NPs, although all were negative, their

absolute z-potentials were apparently higher, conveying

the difference in structures and amount of adsorbed

proteins. It is conceivable that due to the surface negative,

charge adsorption of serum proteins with the same charge

will take more time, so that the protein configuration can

change to achieve the adsorption. As a result of the weaker

binding, during the incubation the surface protein compo-

nents are easily changing too.[33–35] These differences

should be responsible for the faster uptake of the control

and (PAA/PEI)2 coated NPs at the initial stage but after 24h

similar uptake ratios can be observed.

The critical low uptake ratio of the PEGylated NPs can be

explained by the volume exclusion effect of the PEG

molecules, which prevent nonspecific protein adsorption.

Indeed, it has been proved that the apolipoprotein E is not

present on the PEG covered PLA NP surface.[31] z-Potential

measurements confirm the very low adsorption of serum

proteins. The same low adsorption for the serum protein

was observed for the crosslinked particles. The FA

immobilization onto the PEGylated NPs can, however, on

one hand repel the nonspecific protein adsorption, on the

other hand enhance significantly the cellular uptake of the

NPs. Therefore, the PEG-FA grafted NPs can be effectively

delivered to theHepG2cellswhile they retain thegoodanti-

fouling property.

Conclusion

PAA/PEIwere successfully built onpositively charged PLGA

NPs, and can be used for further engineered through

crosslinking and covalent attachment of PEG and PEG-FA

molecules. Thestepwise LbLassemblyandcovalentbinding

of PEG and PEG-FA on the NPs were monitored by z-

potential. The PAA/PEI multilayer coating and PEGylation

provide a simple and stepwise procedure to define the



surface properties of the NPs, especially regarding their

interaction with proteins and cells. Coculture of the

engineered NPs with HepG2 cancer cells revealed that

the cellular uptake of the NPs decreased significantly after

PEGylation.However, ahigher cellularuptakewasobtained

for theNPswhose surfacewas engineeredwith PEG-FA. It is

known that the tumor cells such as HepG2 overexpress the

FRs on theirmembranes, thus the PEG-FA coatedNPs canbe

potentially used as the specific targeting carriers for

anticancer drugs. Our results show that LbL approach

combinedwith covalentbindingof PEGandFAcan tune the

surface properties of the NPs to reduce unspecific interac-

tions and to achieve targeted delivery in cancer cell lines.

Acknowledgements: The authors thank Anderes Pavon fromBioMed institute for support in the use of confocal laser scanningmicroscope. This work is financially supported by the grant MAT2007-60458 from the Spanish Ministry of Science and Innovation,the Natural Science Foundation of China (50873087), the MajorState Basic Research Program of China (2005CB623902), and theNational High-tech Research and Development Program(2006AA03Z442, 2006AA02A140). S. E. Moya is a Ramon y CajalFellow and he thanks this program of the Spanish Ministry ofScience and Innovation for support.

Received: September 23, 2009; Revised: October 28, 2009;Published online: January 7, 2010; DOI: 10.1002/macp.200900514

Keywords: drug delivery system; nanoparticles; poly(ethyleneglycol); poly(lactide-co-glycolide); self-assembly

[1] A. Ambruosi, S. Gelperina, A. Khalansky, S. Tanski, A. Theisen,J. Kreuter, J. Microencapsulation 2006, 23, 582.

[2] L. Costantino, F. Gandolfi, G. Tosi, F. Rivasi, M. A. Vandelli, F.Forni, J. Controlled Release 2005, 108, 84.

[3] J. M. Koziara, P. R. Lockman, D. D. Allen, R. J. Mumper,J. Controlled Release 2004, 99, 259.

[4] E. Allemann, J.-C. Leroux, R. Gurny, E. Doelker, Pharm. Res.1993, 10, 1732.

[5] M. F. Zambaux, F. Bonneaux, R. Gref, P. Maincent, E.Dellacherie, M. J. Alonso, P. Labrude, C. Vigneron,J. Controlled Release 1998, 50, 31.

[6] G. Crotts, H. Sah, T. G. Park, J. Controlled Release 1997, 47, 101.[7] J. Panyam, V. Labhasetwar,Adv. Drug Deliv. Rev. 2003, 55, 329.[8] P. D. Scholes, A. G. A. Coombes, L. Illum, S. S. Daviz, M. Vert,

M. C. Davies, J. Controlled Release 1993, 25, 145.[9] B. Weiss, M. Schneider, L. Muys, S. Taetz, D. Neumann, U. F.

Schaefer, C.-M. Lehr, Bioconjugate Chem. 2007, 18, 1087.[10] S. Izumikawa, S. Yoshioka, Y. Aso, Y. Takeda, J. Controlled

Release 1991, 15, 133.[11] P. Sansdrap, A. J. Moes, Int. J. Pharm. 1993, 98, 157.[12] J. Siepmann, N. Faisant, J. Akiki, J. Richard, J. P. Benoit,

J. Controlled Release 2004, 96, 123.

DOI: 10.1002/macp.200900514


[13] N. Faisant, J. Akiki, F. Siepmann, J. P. Benoit, J. Siepmann, Int. J.Pharm. 2006, 314, 189.

[14] Y. Aso, S. Yoshioka, A. L. W. Po, T. Terao, J. Controlled Release1994, 31, 33.

[15] P. Kocbek, N. Obermajer, M. Cegnar, J. Kos, J. Kristl,J. Controlled Release 2007, 120, 18.

[16] P. A. McCarron, W. M. Marouf, D. J. Quinn, F. Fay, R. E. Burden,S. A. Olwill, C. J. Scott, Bioconjugate Chem. 2008, 19, 1561.

[17] B. Sun, B. Ranganathan, S.-S. Feng, Biomaterials 2008, 29, 475.[18] N. Zhang, C. Chittasupho, C. Duangrat, T. J. Siahaan, C.

Berkland, Bioconjugate Chem. 2008, 19, 145.[19] J. Cheng, B. A. Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E.

Levy-Nissenbaum, A. F. Radovic-Moreno, R. Langer, O. C. Far-okhzad, Biomaterials 2007, 28, 869.

[20] Z. Zhang, S. H. Lee, S.-S. Feng, Biomaterials 2007, 28,1889.

[21] H. Zhao, L. Y. L. Yung, Int. J. Pharm. 2008, 349, 256.[22] S.-W. Choi, J.-H. Kim, J. Controlled Release 2007, 122, 24.[23] C. Sun, R. Sze, M. Zhang, J. Biomed. Mater. Res., A 2006, 78A,

550.[24] F. Sonvico, S. Mornet, S. Vasseur, C. Dubernet, D. Jaillard, J.

Degrouard, J. Hoebeke, E. Duguet, P. Colombo, P. Couvreur,Bioconjugate Chem. 2005, 16, 1181.

[25] G. Decher, Science 1997, 277, 1232.[26] G. Decher, J. D. Hong, Makromol. Chem. Macromol. Symp.

1991, 46, 321.



[27] F. Meng, G. H. M. Engbers, J. Feijen, J. Biomed. Mater. Res., A2004, 70A, 49.

[28] J. Zhou, S. Moya, L. Ma, C. Gao, J. Shen,Macromol. Biosci. 2008,9, 326.

[29] W. Tong, C. Gao, H. Mohwald, Macromolecules 2006, 39, 335.[30] E. Donath, A. Budde, E. Knippel, H. Baumler, Langmuir 1996,

12, 4832.[31] J.-C. Leroux, P. Gravel, L. Balant, B. Volet, B. M. Anner, E.

Allemann, E. Doelker, R. Gurny, J. Biomed. Mater. Res. 1994,28, 471.

[32] J.-C. Leroux, F. D. Jaeghere, B. Anner, E. Doelker, R. Gurny, LifeSci. 1995, 57, 695.

[33] E. Allemann, P. Gravel, J.-C. Leroux, L. Balant, R. Gurny,J. Biomed. Mater. Res., A 1997, 37, 229.

[34] T. Blunk, D. F. Hochstrasser, J.-C. Sanchez, B. W. Muller, R. H.Muller, Electrophoresis 1993, 14, 1382.

[35] T. Blunk, M. Luck, A. Calvor, D. F. Hochstrasser, A. Sanchez,B. W. Muller, R. H. Muller, Eur. J. Pharm. Biopharm. 1996, 42,262.

[36] D. Lechardeur, A. S. Verkman, G. Lukacs, Adv. Drug Deliv. Rev.2005, 57, 755.

[37] H. Mokuno, N. Yamada, H. Shimano, S. Ishibashi, N. Mori, K.Takahashi, T. Oka, T. H. Yoon, F. Takaku, Biochim. Biophys.Acta 1991, 1082, 63.

[38] W. Zauner, N. A. Farrow, A. M. R. Haines, J. Controlled Release2001, 71, 39.


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