Joon Sig Choi et al- Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine

8/3/2019 Joon Sig Choi et al- Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine

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Enhanced transfection efficiency of PAMAM dendrimer by surface

modification with l-arginine

Joon Sig Choia,1, Kihoon Nam b,1, Jong-yeun Park b, Jung-Bin Kimc,Ja-Kyeong Leec, Jong-sang Park b,*

a

Department of Biochemistry, Chungnam National University, Gung-dong 220, Yuseong-gu, Daejeon 305-764, South Korea bSchool of Chemistry and Molecular Engineering, Seoul National University, San 56-1, Shillim-dong, Kwanak-ku, Seoul 151-742, South Koreac Department of Anatomy, Inha University School of Medicine, Inchon, South Korea

Received 16 December 2003; accepted 26 July 2004

Available online 2 September 2004

Abstract

We designed a novel type of arginine-rich dendrimer, with a structure based on the well-defined dendrimer, polyamidoamine

dendrimer (PAMAM). Further characterization was performed to prove that the polymer is a potent nonviral gene delivery

carrier. The primary amines located on the surface of PAMAM were conjugated with l-arginine to generate an l-arginine-

grafted-PAMAM dendrimer (PAMAM-Arg). For comparison, an l-lysine-grafted-PAMAM dendrimer (PAMAM-Lys) was alsogenerated and compared as a control reagent. The polymers were found to self-assemble electrostatically with plasmid DNA,

forming nanometer-scale complexes. From dynamic light scattering experiments, the mean diameter of the polyplexes was

observed to be around 200 nm. We used PicoGreen reagent as an efficient probe for assaying complex formation of polymers

with plasmid DNA. The complex composed of PAMAM-Arg/DNA showed increased gene delivery potency compared to

native PAMAM dendrimer and PAMAM-Lys. The cytotoxicity and transfection efficiencies for 293, HepG2, and Neuro 2A

cells were measured by comparison with PEI and PAMAM. In addition, transfection experiments were performed in primary rat

vascular smooth muscle cells, and PAMAM-Arg showed much enhanced transfection efficiency. These findings suggest that the

l-arginine-grafted-PAMAM dendrimer possesses the potential to be a novel gene delivery carrier for gene therapy.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Dendrimer; l-Arginine; Plasmid DNA; Polyplex; Gene delivery

1. Introduction

The need to develop efficient, reliable, and safe

gene (RNA, DNA) delivery techniques continues to

increase with the development of applications for

gene therapy. During the past decade, intensive

research and development has been carried out in

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2004.07.027

* Corresponding author. Tel.: +82 2 880 6660; fax: +82 2 877

5110.

E-mail address: [email protected] (J. Park).1 The first two authors contributed equally to this work.

Journal of Controlled Release 99 (2004) 445–456

www.elsevier.com/locate/jconrel

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pursuit of effective methods for transferring therapeu-

tic genes into cells with the aim of human gene

therapy. Several clinical trials reported so far have

involved viral vector systems (retroviruses, adenovi-ruses) that provide efficient transduction and high

levels of gene expression. However, there are still

some key safety issues that need to be addressed such

as inherent toxicity, short- and long-term risks such as

the generation of host immune responses, and the

possibility of inserted genes combining to activate

oncogenes. For these reasons, the demand for an

alternative to viral vectors—i.e., nonviral vector

systems—has increased, and several techniques have

emerged that are regarded as safer and more desirable

methods for gene delivery and clinical gene therapy[1,2]. Nonviral vector systems usually make use of

either naked plasmid DNA only or various kinds of

DNA-complexing agent such as cationic liposomes

and polycationic polymers. The inefficiency and

cytotoxicity associated with the synthetic nonviral

systems currently in use should be considered during

in vivo use. Consequently, only a few nonviral vectors

have reached clinical trials.

Among the nonviral vector systems, several

synthetic and natural cationic polymers have been

introduced and tested for their potential applicability

to the field of gene therapy. While some cationic

polymers showed promise during the first stage of

trial, unexpected characteristics such as low trans-

fection efficiencies in vivo and inherent cytotoxicity

eventually limited their use as in vivo gene carriers

[3,4]. Nevertheless, polycationic dendrimers are still

attractive to many scientists because of their well-

defined structure and easy control of surface function-

ality for the design of biomedical applications [5]. At

present, polyamidoamine (PAMAM) dendrimer and

polyethylenimine (PEI) dendrimer have been tested

for their potential utility and have exhibited relativelyhigh transfection efficiencies in vitro [6–9] with PEI

showing some promising results in vivo [10,11].

As described above, one of the major problems with

nonviral gene delivery systems is their lower effi-

ciency compared to viral vectors. Many techniques

have been tried to overcome such problems, including

linking or conjugating cell-specific ligands and TAT-

derived peptide or oligopeptide, such as oligoarginine

derivatives. Recently, some basic peptides known as

protein transduction domains (PTD) or membrane

translocalization signals (MTS) were identified, char-

acterized, and introduced to various therapeutic

applications for the delivery of drugs, proteins,

oligonucleotides, and plasmid DNA [12,13]. Interest-ingly, it is known that these sequences usually contain

positively charged amino acid residues, i.e., arginine

and lysine. Even though the real mechanism is still

unclear and there is debate about whether the entry into

cell membranes follows an endocytic or nonendocytic

pathway or direct penetration into membranes [14], the

phenomenon of enhanced transportation into cells has

been reported by many groups. Most of the experi-

ments were usually performed by covalently linking

nucleic acids to the signal peptides for delivery, and

some trials were also conducted by simple complex-ation by electrostatic interaction.

The focus of this paper is to present a new three-

dimensional artificial protein, l-arginine-grafted-

PAMAM dendrimer (PAMAM-Arg), as a novel

nonviral gene delivery vector, which is composed

of a backbone of PAMAM dendrimer and the

surface of which is covered with the basic l-arginine

residues. Interestingly, by introducing arginine resi-

dues to the dendritic surfaces, gene delivery potency

greatly increased in comparison with that of native

PAMAM and was comparable to PEI for HepG2 and

primary rat vascular smooth muscle cells, and was

more efficient in the case of Neuro 2A cells than

PEI and Lipofectamine. As a control, l-lysine-

grafted-PAMAM (PAMAM-Lys) was prepared and

tested showing slightly better transfection efficiency

in HepG2 cells than that of native PAMAM, while

no increased effect was observed in primary cells.

2. Materials and methods

2.1. Materials

PAMAM G4 (Starburst), 3-[4,5-dimethylthiazol-2-

yl]-2,5-diphenyl tetrazolium bromide (MTT), piper-

idine, N , N -dimethylformamide (DMF), N , N -diisopro-

pylethylamine (DIPEA), and BSA were purchased

from Sigma-Aldrich Korea. N -hydroxybenzotriazole

(HOBt), and 2-(1 H -benzotriazole-1-yl)-1,1,3,3-tetra-

methyluronium (HBTU) were purchased from Anas-

pec (San Jose, CA) and Fmoc-l-Arg(pbf)-OH was

from Novabiochem (San Diego, CA). Luciferase

J.S. Choi et al. / Journal of Controlled Release 99 (2004) 445–456 446



assay kit was from Promega (Madison, WI). Pico-

Green reagent was obtained from Molecular Probes.

Fetal bovine serum (FBS), fetal calf serum (FCS),

100ÂAntibiotic–antimycotic agent and Dulbecco’smodified Eagle’s medium (DMEM) were purchased

from GIBCO (Gaithersburg, MD). DMEM/F-12 was

from Cambrex (Walkersville, MD). Collagenase type

II, elastase, and soybean Trypsin inhibitor were from

Worthington (Lakewood, NJ).

2.2. Synthesis of PAMAM-Arg and PAMAM-Lys

Amino acid coupling to the PAMAM was per-

formed in anhydrous DMF for 4 h at room temper-

ature with 4 equivalents of HOBt, HBTU, Fmoc-Arg(pbf)-OH and 7.3 equiv. of DIPEA, respectively.

The product was precipitated in ethyl ether and

washed with excess ether. The Fmoc groups of

Fmoc-Arg(pbf)-coupled dendrimer was removed by

adding 30% piperidine in DMF (v/v). After 1 h of

deprotection reaction, the mixture was precipitated in

ethyl ether and washed with excess ether. The reagent

(95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/

H2O, v/v) was used for deprotection of pbf groups

of arginine (6 h at room temperature) and the final

product was precipitated in ethyl ether and washed

with excess ether. The product was solubilized in

water and dialyzed against pure water at 4 8C for

overnight. Then, the product was collected after

freeze-drying yielding white powder. The overall

synthesis scheme of PAMAM-Lys was the same as

described above. Fmoc-Lys(Boc)-OH was usedinstead and 90% TFA was used finally to deprotect

BOC groups (1 h at room temperature). The yields for

the products were usually over 99%. The 1H NMR

spectra (300 MHz, D2O) of the polymers are

displayed in Fig. 1.

2.3. Plasmid preparation

The firefly luciferase gene was used as a reporter

gene to monitor the result of gene transfection. The

luciferase expression plasmid (pCN-Luci) was con-structed by subcloning cDNA of Photinus pyralis

luciferase with 21-amino acid nuclear localization

signal from SV40 large T antigen to pCN [15]. pCN

vector was known to express higher than the vector

with only CMV promoter [16]. Plasmid DNA was

transformed into E. coli TOP10 competent cells and

highly purified covalently closed circular plasmid

DNA was isolated by plasmid purification Mega kits

from Qiagen (Valencia, CA, USA) according to the

manufacturer’s instructions. Plasmid was precipitated

in isopropanol and further washed with 70% ethanol

twice and resuspended in distilled water. DNA was

stored at À20 8C until use.

Fig. 1. 1H NMR data of PAMAM, PAMAM-Arg and PAMAM-Lys.


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2.4. Agarose gel electrophoresis studies

Complexes were formed at different charge ratios

between the polymer and pCN-Luciferase (pCN-Luci) plasmid by incubating in HEPES buffer (25 mM, pH

7.4, 10 mM MgCl2) at room temperature for 30 min.

Each sample was then analyzed by electrophoresis on

a 0.7% agarose gel and stained by incubation for 1 h

in buffer containing ethidium bromide (0.5 Ag/ml) at

37 8C.

2.5. PicoGreen assay for polyplex formation

Polyplexes of plasmid DNA (1.0 Ag) and den-

drimers were prepared at various charge ratios,ranging from 0.5 to 30, in 200 Al of HEPES-buffered

saline (HBS, 25 mM HEPES, 150 mM NaCl, pH 7.4),

and the mixtures were incubated for 30 min at room

temperature. Then, 200 Al of PicoGreen reagent

diluted in TE buffer (10 mM Tris, 1 mM EDTA, pH

7.5) was added and incubated further for 2 min. The

polyplexes were diluted to a total of 2 ml of TE buffer

prior to measuring fluorescence intensity with a

spectrofluorometer (JASCO FP-750). Excitation

(kex) and emission (kem) wavelengths were 480 and

520 nm, respectively.

2.6. Zeta potential and particle size measurements

The zeta potential values and size of polyplexes

were determined by the Malvern Zetasizer 3000HAs

system (Malvern Instruments, Worcestershire, UK)

using the PCS 1.61 software. Polyplexes were formed

at a final concentration of 5 Ag/ml plasmid DNA in

HBS (10 mM HEPES, 1 mM NaCl, pH 7.4) for zeta

potential experiments and in water for size measure-

ments, respectively.

2.7. Cell culture

Human embryonic kidney 293 cells and Nuero 2A

(mouse neuroblastoma) cells were grown in DMEM

with 10% FBS. Human liver carcinoma HepG2 cells

were propagated in MEM supplemented with 10%

FBS. The primary rat aorta vascular smooth muscle

cells were grown in DMEM/F12 containing 10%

FBS. The cells were routinely maintained on plastic

tissue culture dishes (Falcon) at 37 8C in a humidified

atmosphere containing 5% CO2/95% air. All media

routinely contained 1Â antibiotic–antimycotic agent.

2.8. Primary rat aorta vascular smooth muscle cells preparation

The cells were isolated from rat aortic vascular

smooth muscle in a primary culture [17,18]. Briefly,

the enzyme mixture containing 1 mg/ml collagenase

type II, 0.25 mg/ml elastase, 1 mg/ml soybean Trypsin

inhibitor, and 2 mg/ml BSA prepared in DMEM/F12

was pre-warmed at 37 8C and incubated with aorta

which was obtained from 6-week-old SD rats and cut

into small pieces (Charles River, Japan). The digestion

was carried out for 30–45 min with stirring at 37 8C.The resulting cell suspension was centrifuged at 1000

rpm for 5 min at 4 8C, and the resulting cell pellet was

washed with DMEM/F12 medium containing 10%

FCS, 100 mg/ml penicillin, and 0.1 mg/ml strepto-

mycin. The pellet was then resuspended in 5 ml of

fresh medium. The cells were then maintained in

DMEM/F12 media containing 10% FBS and 1Â

antibiotic–antimycotic agent. The cells in passage

numbers between 3 and 12 were used for transfection

experiments.

2.9. Cytotoxicity assay in vitro

For the cytotoxicity assay, the colorimetric MTT

assay was performed [19]. Briefly, 293 and HepG2

cells were seeded at a density of 1Â104 cells/well in a

96-well plate and grown in 100 Al of media for 1 day

prior to the incubation with polymers. After treating

cells with PEI 25 kDa, PAMAM G4, PAMAM-Arg

and PAMAM-Lys for 2 days, 26 Al of MTT stock

solution (2 mg/ml) was added to each well and

incubated further for 4 h. The media was removed and

150 Al of DMSO was added and the absorbance wasmeasured at 570 nm using a microplate reader.

2.10. Transfection experiments and assay

293 (5Â104 cells/well), HepG2 (1Â105 cells/well),

and Neuro 2A (1Â105 cells/well) cells were seeded in

24-well plates and grown in 600 Al of medium

containing 10% FBS for a day before transfection.

For primary rat vascular smooth muscle cells, 4Â104

cells/well were seeded and grown for 2 days before




transfection. Polyplexes of plasmid DNA and den-

drimers were prepared at various charge ratios in 150

Al of FBS-free media, and the mixtures were

incubated for 30 min at room temperature. For FBS-free condition, the medium was replaced by 600 Al

FBS-free medium before transfection. Following 4 h

treatment of polyplexes, the medium was replaced by

600 Al medium containing 10% FBS. Cells were

incubated further for 2 days before assay. After the

growth medium was removed, cells were washed with

DPBS and lysed for 30 min at room temperature using

150 Al of Reporter lysis buffer (Promega). Luciferase

activity was measured using a LB 9507 luminometer

(Berthold, Germany) and the protein content was

measured by using a Micro BCA assay reagent kit (Pierce, Rockford, IL).

3. Results and discussion

3.1. Synthesis and features of PAMAM-Arg and

PAMAM-Lys

It was reported that arginine-rich peptides can

serve as effective gene delivery vectors [20–22]. It has

also been shown that oligoarginines (n=6–9) are

membrane translocational signals, and several chi-

meric peptides have been developed during the past

few years [12–14]. Recently, arginine dendrimers

were reported by Kasai et al. [23], who introduced

eight or 16 arginine residues on each primary amino

group of the poly-l-lysine dendrimer backbone and

compared the antiangiogenic activity with poly-l-

lysine dendrimer itself. More recently, Okuda et al.

[24] reported that higher transfection efficiency could

be obtained by replacing the terminal lysines of

dendritic poly(l-lysine) with arginines. The present

study investigates the possibility of creating a potent transfection agent by grafting arginine residues onto

the dendritic surface of the PAMAM dendrimer

(generation 4), which is commercially available at a

relatively low cost, is reasonably small in molecular

weight, and contains a reasonable number of tertiary

amines that are believed to contribute to the endo-

some-buffering effect. To characterize the effects on

the gene delivery efficacy of introducing arginine

residues, which are the key units of the cell-penetrat-

ing peptides, arginines were positioned at each

terminal end of PAMAM by conjugating them to the

surface primary amines of PAMAM dendrimer. The

purpose of the design and synthesis of the dendrimer

is that the surplus of spatially oriented arginineresidues after complex formation with DNA might

contribute to enhanced uptake by cells, resulting in an

increased transfection ef ficiency.

As shown in Fig. 1, the 1H NMR spectra of the

polymers are like as followings. 1H NMR (300 MHz,

D2O) PAMAM: d 2.44 (–NCH2C H 2CO– of PAMAM

unit, 248H), 2.63 (–CONHCH2C H 2 N– of PAMAM

unit, 120H and –NC H 2C H 2 N– of PAMAM unit, 4H),

2.72 (–CONHCH2C H 2 NH2 of PAMAM unit, 128H),

2.83 (–NC H 2CH2CO– of PAMAM unit, 248H), 3.25

(–CONHC H 2CH2 N– of PAMAM unit, 248H).PAMAM-Arg: d 1.68 (–HCCH2C H 2CH2 NH– of

arginine, 116H), 1.93 (–HCC H 2CH2CH2 NH– of argi-

nine, 116H), 2.63 (–NCH2C H 2CO– of PAMAM

u ni t, 2 48 H) , 2 .8 3 ( –C ON HC H2C H 2 N – o f

PAMAM unit and –NC H 2C H 2 N– of PAMAM

unit), 2.96 (–CONHCH2C H 2 NH2 of PAMAM

unit), 3.13 (–NC H 2CH2CO– of PAMAM unit), 3.25

(–HCCH2CH2C H 2 NH– of arginine), 3.46 (–CON

HC H 2CH2 N– of PAMAM unit and –CONHCH2

C H 2 NHCO– of PAMAM unit), 4.01 (– H CCH2

CH2CH2 NH– of arginine, 58H). PAMAM-Lys: d

1.45 (–CHCH2C H 2CH2CH2 NH2 of lysine, 120H),

1 .7 3 ( –C HC H2C H 2 C H 2 N H 2 o f l ys in e,

120H), 1.89 (–CHC H 2CH2CH2 NH2 of lysine,

120H), 2.53 (–NCH2C H 2CO– of PAMAM unit,

2 4 8 H ) , 2 . 8 1 ( – C O N H C H 2 C H 2 N – o f

PAMAM unit, and –NC H 2C H 2 N– of PAMAM

unit), 3.02 (–CONHCH2C H 2 NH2 of PAMAM unit,

–NC H 2CH2CO– of PAMAM unit and –CHCH2

CH2CH2C H 2 NH2 o f l y si n e) , 3 . 39 ( – CO N H

C H 2CH2 N– of PAMAM unit and –CONHCH2

C H 2 NHCO– of PAMAM unit), 3.92 (–C H CH2

CH2CH2CH2 NH2 of lysine, 60H).The number of arginine and lysine residues

attached was calculated from 1H NMR data. Approx-

imately 58 molecules of arginine were coupled to a

single PAMAM dendrimer that contained 64 surface

primary amines, with the number also corresponding

well with the PAMAM-Lys preparation (60 lysines

per PAMAM). To study the possible role of

increased MW and charge density of polymers,

PAMAM-Lys was prepared and compared as a

control agent.


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3.2. Analysis of complex formation by agarose gel

electrophoresis and PicoGreen reagent assay

To assess the formation of dendrimer/DNA complexes, agarose gel electrophoresis of the polyplexes

was performed at different charge ratios (Fig. 2). The

plasmid DNA showed complete retardation at a

charge ratio of two with PAMAM-Arg or PAMAM-

Lys. In addition, for more precise analysis of

complex formation, PicoGreen reagent was used to

assess polyplex formation at various charge ratios. A

commonly used method of analyzing the formation of

polymers/DNA complexes is the ethidium bromide

exclusion assay. However, the method possesses

some problems in that it is critically dependent onthe concentration of ethidium bromide used and is

low in sensitivity, giving only a 10- to 15-fold

enhancement of fluorescence compared to the pos-

itive control. We report here a novel method for the

quantification of complex formation with DNA using

PicoGreen reagent as a more sensitive probe. This

method was found to be highly reproducible and very

sensitive for monitoring complex formation. Prior to

the addition of buffer containing an appropriate

amount of PicoGreen reagent, the polymer was

allowed to self-assemble with plasmid DNA by

incubating for 30 min at room temperature. The

uncomplexed part of the plasmid DNA is exposed to

the PicoGreen reagent, and the resultant fluorescence

increases according to the degree of uncomplexation.

As presented in Fig. 3A, the inhibition of fluores-

cence resulting from precomplexation with the

dendrimers increased with increasing charge ratios,

reaching a plateau near zero at around a charge ratio

of 4. This means that plasmid DNA was condensed

into particles so efficiently that no DNA could

Fig. 2. Agarose gel electrophoresis retardation assay of plasmid DNA by PAMAM-Arg (A) and PAMAM-Lys (B). Plasmid DNA (0.4 Ag) only

(lane 1); charge ratio of polymer/DNA=0.5, 1, 2, 4 and 8 (lanes 2, 3, 4, 5 and 6, respectively).

Fig. 3. (A) The DNA complexation assay of polymers using

PicoGreen reagent at various charge ratios. After formation of

complexes with DNA, PicoGreen reagent was added and fluo-

rescence (ex 480 nm, em 520 nm) was measured at each condition.

PEI (.), PAMAM (E), PAMAM-Arg (n), PAMAM-Lys (z) and

PAMAM-OH(4). (The asterisk for PAMAM-OH means the weight

ratio of polymer to DNA.) (B) The zeta potential values of polymer/

DNA complexes at various charge ratios. PAMAM (E), PAMAM-

Arg (n), and PAMAM-Lys (z). Results are expressed as mean-

Fstandard deviation (n=3).




interact with the added PicoGreen reagent. Interest-

ingly, it was observed that native PAMAM showed

complete complex formation at a charge ratio of 2,

which was lower than other polymers that required acharge ratio of at least 4. The results show that native

PAMAM was more effective at forming complete

complexes, and PAMAM-Arg and PAMAM-Lys

could effectively form condensed complexes not

unlike PEI and PAMAM at a charge ratio of 4. It is

thought that the surface of the native PAMAM is

covered with primary amines except that they are

higher in p K a value when compared with the a-

amines of lysines or arginines and the secondary or

tertiary amines of PEI, which were included in

calculating the charge ratios, respectively. As acontrol experiment, PAMAM-OH polymer that could

not form polyionic complexes with DNA was also

tested, and it was observed that the relative fluo-

rescence remained around 100% at all the weight

ratios tested.

3.3. Zeta potential and size measurements of the

polyplexes with plasmid DNA

From the above agarose gel retardation and Pico-

Green assay results, it was presumed that the modified

dendrimers, PAMAM-Arg and PAMAM-Lys, could

form compact polyplexes. The surface charges of the

complexes were measured and displayed in Fig. 3B.

In accordance with the previous profile obtained in the

charge ratio-based PicoGreen assay curve in Fig. 3A,

unmodified PAMAM showed that the value was 20

mV at a charge ratio of 2 in comparison with other

c om pl ex es c om po se d o f D NA a n d m od if ie d

PAMAM-Arg or PAMAM-Lys, both of which dis-

played positive values below 10 mV at the same

charge ratio. In addition, the zeta potential measure-

ments of all the polyplexes displayed equivalent

values that were around 20–25 mV at charge ratiosof 4 or greater.

The formation of complexes at the nanometer

level is generally considered to be one of the

important factors in polyplex-mediated gene delivery.

The mean particle size of polyplexes was examined

by dynamic laser light scattering. As shown in Table

1, the polymers efficiently condensed DNA into

nanometer-sized particles with sizes ranging from

185 to 250 nm at their optimal transfection con-

ditions. Interestingly, the mean size of polyplexes

composed of PAMAM and DNA was 245 nm, whichwas slightly larger than those of PAMAM-Arg/DNA,

PAMAM-Lys/DNA, and PEI/DNA complexes with

values around 200 nm. Based on the previous

PicoGreen reagent assay results and the results in

Table 1 showing that the charge density per modified

dendrimer increased by 16% and 25% for PAMAM-

Arg and PAMAM-Lys, respectively, compared to

native PAMAM, it is considered that the modified

polymers also could fully compensate for the

phosphate anions of plasmid DNA, and they could

form mature polyplexes much like native PAMAM or

PEI. Therefore, at a charge ratio of 6, which was

found to be sufficient for the formation of complete

complexes, PAMAM-Arg and PAMAM-Lys could

form complexes that exposed multiple surplus sur-

face arginine or lysine residues. In addition, no

visible precipitation due to the increase in size was

detected even when 150 Ag /m l o f D NA w as

complexed with PAMAM-Arg in pure water or 5%

glucose solution.

Table 1

The comparison of the physicochemical characteristics of the polymers and size measurements of the complexes with plasmid DNA

PEI PAMAM PAMAM-Arg PAMAM-Lys

MW (Da) 25 000a 14215a 23321 b 21895 b

No. of (+)/polymer 581 64 122 124

No. of (+)/1 Ag 1.3990Â1016 2.7104Â1015 3.1493Â1015 3.4094Â1015

Mean size (nm)c 208.5F1.5 246.4F1.5 200.2F2.6 185.1F0.5

a Molecular weight as provided by the manufacturers. b Molecular weight as determined by 1H NMR analysis of each polymer.c Mean size as measured by dynamic light scattering experiments and meanFstandard deviations are given (n=3). The charge ratios (N/P)

of complexes were 7.8 for PEI and 6.0 for PAMAM, PAMAM-Arg, and PAMAM-Lys.


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3.4. Cytotoxicity issues

The toxicity of cationic polymers was reported to

be a function of the interactions of the polymers withcell membranes and/or of the efficiency of cellular

uptake [25,26]. We recently reported that quaternized

PAMAM-OH derivatives showed a lower level of

cytotoxicity than PAMAM because they maintain

cationic charges inside the polymeric back bone

shielded by surface hydroxyl groups [27]. As

presented in Fig. 4, we compared the cytotoxicity

of the reagents on 293 and HepG2 cells. Each cell

was incubated for 48 h with increasing amounts of

polymers in the presence of serum. From the results,

we observed that PEI was highly toxic to both cells

and PAMAM was much less toxic. Both PAMAM-Arg and PAMAM-Lys showed slightly increased

toxicity compared to native PAMAM. This was

expected to be due to the increased charge density

and molecular weight of each modified polymer.

However, the cytotoxicity of both PAMAM-Arg and

PAMAM-Lys showed much lower levels compared

with that of PEI. We presumed that if PAMAM-Arg

or PAMAM-Lys could exert a higher level of

transfection than, or at least as much as, that of

PEI, the polymers should be more adequate and

promising vectors for possible in vivo gene deliveryapplications.

3.5. Transfection efficiency on cell lines

Arginine-oligopeptides modified with several

hydrophobic lipids have been recently reported to be

effective gene carriers and, interestingly, those pep-

tides alone did not show a high level of transfection

efficacy [28]. In addition, TAT–PEG–PE liposomal

systems encapsulating plasmid DNA have been

reported to be efficiently incorporated into cells in

vitro and in vivo [29,30]. The common characteristic

of those systems is thought to be that the arginine

residues are rich on the surface of multivalent

liposomal systems. From these reports, we deduced

that if arginine residues could be located on the

surface of PAMAM dendrimer, the change in charac-

teristics might be pronounced as the recent report

about arginine-grafted poly(l-lysine) dendrimer [24].

It was also reported that branched-chain arginine

peptides showed a different cellular localization and

implied that a linear structure was not necessary, and

forming a cluster of arginines was suggested to beimportant for translocation [14].

For a basic experiment, we chose 293 cells first

because the cells are usually vulnerable to conven-

tional nonviral transfection agents. As shown in Fig.

5A, the transfection efficiency of PAMAM, PAMAM-

Arg, and PAMAM-Lys exhibited essentially the same

efficiency as that of PEI whether in the presence of

serum or not. There were no direct observations

regarding whether grafting arginines contributed to

gene delivery potency or not. To further characterize

Fig. 4. Cytotoxicity assay in 293 (A) and HepG2 (B) cells by MTT

assay. PEI (.), PAMAM (E), PAMAM-Arg (n) and PAMAM-Lys

(z). Relative cell viability was calculated as 100Â[( A570 of

polymer-treated cellsÀ A570 of blank)/( A570 of control cellsÀ A570

of blank)]. Each data point represents the meanFstandard deviation

(n=6).




this, HepG2 cells were chosen for the next step

because the transfection efficiency of native PAMAM

(G4) is usually over 10-fold less than that of PEI. In

Fig. 5B, the results were shown for HepG2 cells in the

presence or absence of serum at the optimal con-ditions for each polymer. We observed that the

advantage of the PAMAM-Arg preparation over the

unmodified PAMAM was pronounced (more than one

order of magnitude) and comparable to PEI. In

addition, it could also be noticed that the increased

MW and charge density produced by grafting basic

amino acids to PAMAM also contributed to trans-

fection potency from the PAMAM-Lys results. How-

ever, the effect showed only a small increase in the

level of efficiency compared with that of native

PAMAM. To characterize this further, transfection

experiments were performed and compared at various

charge ratios for PAMAM-Arg, PAMAM-Lys, and

PAMAM. For each charge ratio tested (charge ratio 1–

12), a significant improvement in transfection effi-ciency was observed with PAMAM-Arg compared to

PAMAM-Lys and unmodified PAMAM (Fig. 5D). In

addition, for possible gene delivery applications to

neuronal cells, Neuro 2A cells (mouse albino neuro-

blastoma) were tested in further transfection experi-

ments. To compare the transfection efficiency and

cytotoxicity of each complex, two different concen-

trations of DNA (0.2 and 1.0 Ag/well) were applied

with the results being more pronounced at these

concentrations. As presented in Fig. 6A, PAMAM-

Fig. 5. Transfection efficiency in different cell lines. Each data point represents the meanFstandard deviation (n=3). (A) 293 cells, 1 Ag DNA/ well, black bars for (À) FBS, gray bars for (+) FBS condition. (B) HepG2 cells, 1 Ag DNA/well, black bars for (À) FBS, gray bars for (+) FBS.

(C) DNA dose dependence on the luciferase gene expression for HepG2 cells in the presence of 10% FBS. DNA amount per well was 0.5 Ag

(black), 1.0 Ag (gray) and 2.0 Ag (white). (D) Comparison of transfection efficiency at various charge ratios for HepG2 cells at 2.0 Ag DNA/

well in the presence of serum between PAMAM (E), PAMAM-Arg (n) and PAMAM-Lys (z). Numbers in parentheses represent charge ratios

(+/ À).


G E N

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D E L I V E R Y



Arg produced the highest gene expression level

compared with other reagents. The transfection

efficiency of PAMAM-Arg was more than two ordersof magnitude greater compared with Lipofectamine or

native PAMAM, and more than one order of

magnitude greater compared with PEI. The cytotox-

icity test was also performed for the polyplexes, and

the results are presented in Fig. 6B. At the concen-

tration of DNA used, significant toxicity was

observed for Lipofectamine/DNA complexes (67%

and 53% viability for 0.2 and 1.0 Ag DNA/well,

respectively), whereas other polymer/DNA complexes

showed negligible levels of toxicity at both concen-

trations. Our results, which agree with another recent

observation [24], clearly showed that the transfection

efficiency was significantly increased by introducing

arginines onto the surface of the PAMAM dendrimer.Taken together, the results with HepG2 and Neuro 2A

cells indicate that the arginine-grafted PAMAM

increased transfection efficacy remarkably and was

reproducible.

The results show that PAMAM-Arg increased

gene transfection efficiency in HepG2 and Neuro

2A cells under the action of surface-grafted arginine

residues. Although the actual mechanism still needs

further study, in viewing the zeta potential values of

the complexes in Fig. 3B, the surface charge-based

adsorption of the complexes to cell membranesrevealed no significant difference among the poly-

mers. That is, the difference in the degree of

charge-based interaction with the cell membranes

could not explain the increased gene expression

level of PAMAM-Arg compared with PAMAM and

PAMAM-Lys. Therefore, it is presumed that the

increased gene expression might be attributed to the

difference in either the cell-penetrating activity

during uptake or nuclear localizing efficiency after

entry into the cytosol of the affluent arginine

residues oriented on the surface of PAMAM-Arg/

DNA complexes, or to the synchronous function of

both effects.

3.6. Transfection efficiency for primary rat vascular

smooth muscle cells

Based on previous observations, further experi-

ments were performed for primary rat aorta vascular

smooth muscle cells. These cells were reported to be

related to the renarrowing or restenosis of the artery

after coronary intervention of an intravascular

scaffolding device, known as a stent, to patientssuffering from coronary artery disease (stenosis)

[31]. Although the mechanisms of restenosis are

only partially understood, it is evident that release of

platelet-derived growth factor (PDGF) promotes

smooth muscle cell proliferation and migration to

the injury site. Eventually, these cells contribute to

thrombus formation, which is one of the reasons for

restenosis. Therefore, it was believed to be important

to obtain a high level of gene transfection efficiency

with PAMAM-Arg for the cells in order to establish

Fig. 6. Transfection efficiency for Neuro 2A cell lines (1Â105 cells/

well) and cytotoxicity assay of each complex. DNA amount per well

was 0.2 Ag (black) and 1.0 Ag (gray). (A) The luciferase expression

mediated by reagents was measured at each optimum condition and

presented. (B) Each complex with DNA was incubated with the

cells for 24 h. After replacement with fresh medium, MTT assay

was performed. Results are expressed as meanFstandard deviation

(n=3).




a possible DNA-based gene therapy protocol for the

prevention of restenosis. As demonstrated in Fig. 7A,

PAMAM-Arg showed a significant increase in

potency compared with native PAMAM andPAMAM-Lys, and the efficiency was almost com-

parable to that of PEI in the absence or presence of

serum. The DNA dose dependence of luciferase

expression by the vector was performed and shown

in Fig. 7B. The expression increased as the amount

of DNA introduced increased in the absence of

serum. However, the expression level remained at a

lower level even though the DNA dose increased in

the presence of serum. From these results, it was

observed that the transfection mediated by PAMAM-

Arg of rat aorta smooth muscle cells is hampered by

the presence of serum.

4. Conclusions

In summary, we have described the development of

surface-modified PAMAM derivatives with arginines

or lysines, which were named PAMAM-Arg and

PAMAM-Lys, respectively. PAMAM-Arg showed

enhanced gene expression in HepG2 and Neuro 2A

cell lines and for primary rat vascular smooth muscle

cells in comparison with native PAMAM and

PAMAM-Lys. This constitutes a subnanosized three-

dimensional and multivalent arginine multimer, which possesses the potential to be an efficient gene carrier.

The above results lead us to conclude that the

outstanding transfection efficiency with relatively

low cytotoxicity and ease of preparation would make

PAMAM-Arg a promising nonviral vector for both in

vitro and in vivo use. Potentially, PAMAM-Arg could

be used as a dendritic carrier molecule and could

encapsulate or entangle cargo molecules such as small

molecules, peptides, proteins, oligonucleotides, and

plasmids that are deficient in cell-penetrating or

plasma membrane crossing capability.

Acknowledgement

This work was supported by grants from the

Research Center for Molecular Therapy at Sung-

KyunKwan University, the Korea Science and Engi-

neering Foundation (R02-2002-000-00011-0), and the

Korea Research Foundation (2001-015-DP0344).

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Fig. 7. Transfection efficiency for primary rat aorta smooth muscle

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Joon Sig Choi et al- Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine