tae-il kim et al- pamam-peg-pamam: novel triblock copolymer as a biocompatible and efficient gene...

8/3/2019 Tae-il Kim et al- PAMAM-PEG-PAMAM: Novel Triblock Copolymer as a Biocompatible and Efficient Gene Delivery Carrier

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PAMAM-PEG-PAMAM: Novel Triblock Copolymer as aBiocompatible and Efficient Gene Delivery Carrier

Tae-il Kim, Hyo Jung Seo, Joon Sig Choi, Hyung-Suk Jang, Jung-un Baek,

Kwan Kim, and Jong-Sang Park*,

School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shillim-dong,Kwanak-gu, Seoul 151-742, Korea, and Department of Biochemistry, Chungnam National University,

220 Gung-dong, Yuseong-gu, Daejeon 305-764, Korea

Received July 30, 2004

A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene

carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain

problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer

a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-

solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly

with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our

expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed

little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable

to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to

create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.

Introduction

Many types of dendrimers have been designed and utilized

in many applications, including chemistry and pharmaceu-

tics.1-3 For gene delivery systems, the development of

efficient and nontoxic gene carriers is the most demanding

task, and hundreds of polymeric gene carriers have been

manufactured in numerous laboratories around the world.4

Among them, poly(amidoamine) (PAMAM) dendrimer is

identified as an efficient carrier, and its fractured form, Super-

fect, is commercialized as a gene delivery carrier showing

high transfection efficiency comparable to that of polyeth-

ylenimine (PEI), one of the most efficient gene delivery

carriers in existence.5-7 However, PAMAM dendrimer is still

associated with several problems, including low water

solubility and cytotoxicity, which need to be overcome for

in vivo applications.8 As a consequence, there have been

many trials in which poly(ethylene glycol) (PEG) was linked

to PAMAM dendrimer in order to resolve these issues. 9,10

Since PEG exhibits such properties as nonimmunogenecity,

biocompatibility, and improved water solubility, it has been

coupled to various polymeric gene delivery carriers.11

At first sight, trees may look like AB-type block copoly-mers where the A part constitutes the branch part and the B

part is the trunk of a tree. However, considering their roots,

they are in fact similar to ABA-type copolymers where the

dendritic roots constitute the other A part. So, we tried to

mimic such a simple natural model of multifunctionality and

to engineer hybrid block copolymers for self-assembly with

DNA, which might have potential for gene delivery systems.

A series of PEG-conjugated dendrimers were developed in

our laboratory over the past few years, but these showed

only small transfection activity.12-14 Therefore, we cautiously

expect that this novel copolymer, with its tree-like structure,

would be a more advanced gene delivery carrier.

We report herein the synthesis of the novel triblock

copolymer, poly(amidoamine)-block-poly(ethylene glycol)-

block-poly(amidoamine) (PAMAM-PEG-PAMAM), the phys-

icochemical characterization of complexes formed withDNA, improved properties compared with PAMAM den-

drimer G 4 to which copolymer G 5 corresponds structurally,

and finally the potential of the copolymer for use as a gene

delivery carrier.

Experimental Section

Materials. Poly(ethylene glycol)-bis-amine (MW 3400)

was purchased from Shearwater Polymers (Huntsville, AL).

Bis-amine activity of PEG-(amine)2 was certified to 99%

by the company and PEG was used without further purifica-

tion. Methyl acrylate, ethylenediamine, poly(ethylenimine)

(25kDa), PAMAM dendrimer (Generation 4 solution), and3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) were purchased from Sigma-Aldrich (St. Louis, MO).

pGL3-control vector (5256 bp) was purchased from Promega

(Madison, WI). Fetal bovine serum (FBS), Minimal Essential

Medium (MEM), and Dulbecos modified Eagles medium

(DMEM) were purchased from GIBCO (Gaithersburg, MD).

PAMAM dendrimer was used after evaporation of solution

followed by lyophilization for removal of solvent.

Synthesis of the Copolymer. PEG was used as the

polymeric supporter and the PAMAM dendrimer was

* To whom correspondence should be addressed. Phone: 82-2-880-6660.Fax: 82-2-877-5110.

Seoul National University. Chungnam National University.

2487Biomacromolecules 2004, 5, 2487-2492

10.1021/bm049563j CCC: $27.50 2004 American Chemical SocietyPublished on Web 10/15/2004


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extended outward from the PEG core by repetition of

Michael addition and amidation (Scheme 1).

(1) Michael Addition. First, PEG was dissolved in

methanol and added dropwise to 200 equiv of methyl acrylate

kept at 37 C for the complete reaction. After 48 h, methanol

and unreacted methyl acrylate were removed under vacuum.

The residue was precipitated with an excess of cold ethyl

ether to remove residual methyl acrylate and dried under

vacuum to remove ethyl ether, leaving a white solid,

PAMAM-PEG-PAMAM G 0.5.

(2) Amidation. Second, PAMAM-PEG-PAMAM G 0.5

was dissolved in methanol and added dropwise to 400 equiv

of ethylenediamine kept at 37 C. After 48 h, methanol and

ethylenediamine were removed under vaccum. The residue

was identically precipitated with an excess of ethyl ether to

remove residual ethylenediamine and dried under vacuum

to remove ethyl ether, leaving a weak yellow solid, PAMAM-PEG-PAMAM G 1.0.

These Michael addition and amidation reactions were

performed three or four times repeatedly for the synthesis

of the fourth or fifth generation of the dendritic copolymer.

The polymer was dialyzed for 1 day against ultrapure water

using Spectra/Por dialysis membrane (molecular weight

cutoff) 3500, Spectrum, Los Angeles, CA) after the second

generation, only at full generation to avoid any cleavages of

the ester bonds of the half-generation polymer, and lyoph-

ilized before use for analysis and assay.1H NMR Spectroscopy. 1H NMR spectra of the polymers

were obtained using a Bruker DPX-300 NMR spectrometer

(300 MHz). For analysis, the polymer samples were dissolved

either in MeOD or D2O containing 0.1% and 0.05%

3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt, re-

spectively, as an internal reference (0 ppm).

Gel Retardation Assays. PAMAM-PEG-PAMAM/plas-

mid complexes at various N/P (1, 3 amine groups of

polymer/phosphates of plasmid DNA) ratios ranging from

0.25 to 4.0, were prepared in Hepes buffered saline (HBS,

20 mM Hepes, 150 mM NaCl, pH 7.4). After 30 minincubation at room temperature for complex formation, the

samples were electrophoresed on a 0.7% (w/v) agarose gel

containing ethidium bromide (0.5 g/mL in the gel) andanalyzed on a UV illuminator to show the location of DNA.

Polyplex Stability Measurements in Water. The stability

of polyplex was evaluated by the method developed by

Wadhwa et al. with a slight modification.15 The PAMAM

G 4 dendrimer was used as a control. Each polymer was

mixed with 40 g of plasmid DNA at a constant N/P ratioof 4.0 in 0.4 mL of 20 mM Hepes buffer. After incubation

for 30 min at room temperature, each mixture was centri-

fuged for 5 min at 13000 rpm, 10 C. Then, absorbance of

each supernatant was measured at 260 nm. The stability wascalculated as a percentage ratio of the absorbance of pure

DNA solution and each supernatant.

Atomic Force Microscopy (AFM). The shapes and

particle sizes of the polymer/plasmid DNA complexes were

analyzed using atomic force microscopy (Nanoscope IIIa sys-

tem, Digital Instruments, Inc., Santa Babara. CA). The sam-

ples were prepared by mixing 0.1 g of plasmid DNA withaqueous polymer solution at various N/P ratios to obtain a

final DNA concentration of 10 ng/L. The image of onlyDNA was obtained from the DNA solution containing 40

mM Hepes buffer and 10 mM MgCl2. After 30 min incuba-

tion, 1 L aliquots of the complex solutions were placed ona freshly cleaved untreated mica surface and allowed to stick

for 12 min. Excess solution was removed by careful

absorption onto filter paper and the mica surface was further

dried at room temperature for 24 h. The image mode was

set to tapping mode and the scanning speed was 15 Hz.

Dynamic Light Scattering (DLS). Particle sizes of the

copolymer/plasmid DNA complexes were measured by using

dynamic light scattering. Light scattering experiments were

performed at 25 C with a BI-200SM. goniometer (Brook-

haven Instruments Corporation, Holtsville, NY) using a Lexel

laser (Fremont, CA) model 95 argon laser (100 mW output

power at a wavelength of 514.5 nm). The correlator was

PD2000 (Precision Detectors, Inc., Bellingham, MA) and thescattering angle was 90. Complexes were prepared as

mentioned above, but to final DNA concentrations of 25 g/mL in 1 mL aliquots at various N/P ratios from 0.2 to 18.

All samples were gently inverted for homogenization before

measurement. The particle sizes were measured based on

their average diameters.

Cytotoxicity Assay. Cytotoxicity assay was performed by

the modified MTT assay.16 293 cells were seeded in a 96-

well tissue culture plate at 104 cells per well in 95 L DMEMmedium containing 10% FBS. Cells achieving 7080%

confluence after 24 h were exposed to 5 L of the polymersolutions having various concentrations for 4 h. Cells were

Scheme 1. Synthesis Scheme of PAMAM-PEG-PAMAM: (I)Methyl Acrylate(CH2dCHC(O)OCH3), 37 C, 2 day; (II)Ethylenediamine(H2NCH2CH2NH2), 37 C, 2 Day

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plexes. Copolymer G 5 could retard the migration of DNA

completely even at an N/P ratio of 1, although G 4 could

not retard polyplex until the N/P ratio of the polyplex reached

4. Copolymer G 4 has a nitrogen density of 164.2 Da/N atom

and G 5 has a density of 138.2 Da/N atom theoretically

(Nitrogen density is calculated from the ratio between the

molar mass of the copolymer and the number of nitrogen

atoms). Although copolymer G 5 only exhibits a 1.2-fold

increase in nitrogen density compared to G 4, G 5 has twice

the number (64) of surface primary amines as G 4 (32). This

dramatic retardation result suggests that it is almost certainly

the number of surface primary amines of the copolymer that

is more important for self-assembly with DNA than just

nitrogen density.20 That is to say, internal tertiary amines of

the copolymer could not take part in DNA condensing

because of steric hindrance.

Stability of the Polyplex in Water. Water-stability of the

polyplex is one of the important properties for its in vivo

applications. It is well-known that the polyplexes of cationic

polymers and DNA become hydrophobic, their hydrophilicity

gets lower, and they have a tendency to form insolubleaggregates in water.21 So, we compared the stability of

PAMAM G 4 dendrimer and that of copolymer G 5

polyplexes to verify the role of PEG with respect to polyplex

stability in water. After centrifugation of each polyplex, we

measured the DNA amount of each supernatant at 260 nm

that was formed at a constant N/P ratio of 4. Although the

polyplex of PAMAM G 4 dendrimer exhibits 55% stability,

that of the copolymer shows a value of 88%, if the stability

of DNA alone is regarded as 100% (Figure 5).

This result shows that PEG linked to the copolymer

enhances the colloidal stability of the polyplex considerably.22

From this, we suggest that the surface of the hydrophobic

polyplex core, formed by PAMAM dendrimer blocks of the

copolymer and DNA, is surrounded by hydrophilic PEG

blocks (Figure 6), which corresponds to the previous study

of poly(L-lysine) dendrimer-b-poly(ethylene gycol)-b- poly-

(L-lysine) dendrimer (PLLD-PEG-PLLD) reported by our

laboratory.12

Morphology of the Polyplex. The morphology of the

polyplex of copolymer G 5 and plasmid DNA was observedby AFM. Figure 7A shows the images of only DNA. We

could not observe compact particles at an N/P ratio of 1.2

(Figure 7B). Instead, it was observed that the circular strands

of DNA covered the spherical polyplex core (200 nm

diameter) and stretched outward like a corona. Moreover,

some polyplexes were linked with DNA strands, and they

were shown to form ternary network structures of about

500600 nm in size. Although the DNA was not completely

condensed, it is thought that the retardation of DNA at an

N/P ratio of 1.0 in gel electrophoresis may be due to the net

positive charge of the polyplexes. At an N/P ratio of 5, nearly

all compact polyplexes formed roughly spherical particles

Table 1. Theoretically Calculated and Experimentally Obtained Ratio between Proton (c) and PEG Methylene Proton of the Full GenerationCopolymer, and Molecular Weightsa

polymer generation theoretical # of proton (c) theor. ratio exp. ratio theo. MW Mn Mw Mz PDI

PEG-(amine)2 3327 3369 3423 3475 1.02

G3 56 0.180 0.172 6640 6474 6519 6563 1.01

G4 120 0.386 0.349 10256 9235 9289 9341 1.01

G5 248 0.797 0.722 17488 NDb ND ND ND

a Each molecular weight and PDI were estimated by MALDI-TOF. b ND: not detected. MALDI-TOF MS was not obtained for copolymer G 5 at variousattempted conditions.

Figure 3. MALDI-TOF mass spectra of PEG-(amine)2 (MW 3400).

Figure 4. Gel retardation of (A) copolymer G 4/DNA, (B) G 5/DNApolyplexes. (1) N/P ratio: 0.25, (2) 0.5, (3) 1, (4) 2, (5) 4. C: plasmidDNA only.

Figure 5. Stability measurement of PAMAM-PEG-PAMAM G 5 in

water. All polyplexes were prepared at N/P ratio of 4.0. PAMAM G 4was compared as a control reagent (n)3).

Figure 6. Schematic view of the formation of PEG-coated polyplexes.



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of 100200 nm in size in correspondence with the resultsreported previously (Figure 7C).12

Size of the Polyplex. The sizes and size distribution of

polyplexes were determined by DLS. The copolymer G 5

was used to form polyplexes with DNA. Polyplexes were

prepared at various N/P ratios ranging from 0.23 to 18.4

(Figure 8A). Interestingly, the average size of the polyplex

abruptly increased to 557.3 nm at an N/P ratio of 1.4,

decreased rapidly to 108.6 nm at an N/P ratio of 2.3, and

remained around 170250 nm at N/P ratios from 4.6 to 18.

The abrupt increase of polyplex size at an N/P ratio of 1.4

could be explained that the net charge neutralization of the

polyplex at that N/P ratio induces a hydrophobic interaction

between polyplexes, forming large aggregates in water.18

These size measurement results agreed well with the AFM

results. Figure 8B shows the size distribution of polyplexes

at an N/P ratio of 2.3. The polyplex was found to have a

narrow size distribution, showing homogeneous polyplex

formation. So, we have confirmed that our copolymer formed

nanosized particles with DNA and the average diameter of

the complexes falls within the general size requirements for

efficient cellular endocytosis.

Cytotoxicity of the Copolymer. The cytotoxicity of a

gene delivery carrier is a very important feature. Therefore,

the cytotoxicity of the copolymer G 5 was examined by MTT

assay. 293 transformed human kidney cells were used for

the experiment. As shown in Figure 9, the viability of the

293 cells decreased abruptly with increasing concentration

of PEI. Cytotoxicity was also observed to some extent for

PAMAM G 4 (71% of cells remaining viable) in the 293

cells. However, the copolymer showed little toxicity even

at higher concentration ranges (94% cell viability at 150 g/mL). Generally, the biodegradability of a polymer is known

to decrease its cytotoxicity.23 Interestingly, PEG connected

to the copolymer is thought to inhibit its interaction with

cellular components and so reduce the cytotoxicity of the

copolymer despite its negligibly degradable bonds, which

also corresponds to our previous report.12 This result

demonstrates the safety and biocompatibility of the copoly-

mer and suggests its potential as a gene delivery carrier for

further in vivo applications.

Transfection Efficiency of the Copolymer. The trans-

fection of the copolymer was performed in HepG2 hepato-

cellular carcinoma and 293 transformed human kidney cells

in the absence of chloroquine, which is known to buffer

endosome acidification and eventually result in the release

of internalized polyplex, to examine the polymers ownability. The transfection efficiency (TE) was evaluated by

luciferase reporter gene assay using pGL3 plasmid DNA at

various N/P ratios without serum (Figure 10). For compari-

son, PLLD-PEG-PLLD (G4) polymer was tested together.

Even though the TE of the copolymer for HepG2 was

orders of magnitude less than that of PEI, it was only 30%

less than that of PEI for the 293 cells and comparable to

that of PAMAM G 4 for both cells. Particularly, the

copolymer showed a greatly enhanced TE than PLLD-PEG-

PLLD which is thought to lack endosome buffering effect

like poly(L-lysine) and not be able to escape from endosome

efficiently after endocytosis of the polyplexes, leading a low

Figure 7. AFM images of the copolymer G 5/DNA complexes. (A)plasmid DNA only (B) At N/P ratio of 1.2. (C) At N/P ratio of 5. Thescale bars represent 200 nm.

Figure 8. (A) Average size distribution measurement of PAMAM-PEG-PAMAM G 5 polyplex by DLS. Final DNA concentration is 25g/mL in one milliliter aliquots in various N/P ratios ranging from 0.2to 18. Each data bar shows the average ( standard deviation (n)5). (B) Average size distribution of the polyplex at N/P ratio of 2.3.

Figure 9. Cytotoxicity in 293 cells by MTT assay.

PAMAM-PEG-PAMAM Biomacromolecules, Vol. 5, No. 6, 2004 2491


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TE. Therefore, this enhanced TE of the copolymer may be

due to its own endosome buffering effect like PAMAM

dendrimer. We have verified that the newly synthesized

copolymer showed a relatively high TE.

Generally, polyplexes of cationic gene delivery carriers

are known to interact with negatively charged serum proteins,

which reduce the TE of the carrier acutely.24 So, the TE of

gene delivery carriers should remain unaffected by the pres-

ence of serum for in vivo applications. We compared the

ratio of the TE of each polymer both in the presence and

absence of the serum (with serum/without serum), to examine

the influence of serum presence on the TE (Figure 11).

The ratio of the TEs of PAMAM G 4 was 0.58 at the

optimal N/P ratio, showing greatly decreased TE in the pres-

ence of serum. However, the ratio of the copolymer was 0.85

at an N/P ratio of 14 and was 1.3 even at higher N/P ratio.

This result certainly shows that PEG connected to the copoly-

mer inhibits the interaction of polyplexes with serum protein

and its transfection is not significantly affected by serum

proteins. Moreover, it is our supposition that the polyplex

having a perfect and stable structure formed at a higher ratiowould show greater stability in the presence of serum.

Therefore, the copolymer is found to show more unaffected

TE and undergo a more stable transfection pathway than

intact PAMAM G 4 in the presence of serum. The minimal

impact of serum on the transfection efficiency of PAMAM-

PEG-PAMAM is expected to confer an advantage on the

copolymer for in vivo as well as in vitro gene delivery.

Conclusions

In conclusion, we synthesized a novel tree-like ABA-type

cationic triblock copolymer and investigated its potential as

a gene delivery carrier. The copolymer is composed of a

PEG core and two PAMAM substructures on both sides. It

was able to self-assemble with plasmid DNA forming a

compact polyplex, which showed greatly enhanced water-

solubility compare to the PAMAM dendrimer itself. The

polyplex thus formed was found to have a spherical shape

with a nanosize appropriate to gene delivery and a narrow

size distribution. The copolymer is proven to have little

cytotoxicity in mammalian cells, low interaction with serum,and high transfection efficiency comparable to that of PEI

in 293 cells. Moreover, it showed greatly enhanced TE than

PLLD-PEG-PLLD, which satisfies our expectations. There-

fore, it has the potential to become one of biocompatible

and efficient gene delivery carriers.

Acknowledgment. We thank Kihwan Choi for doing the

DLS measurements. This work was supported by Korea

Science and Engineering Foundation (R02-2002-000-00011-

0), Korea Research Foundation (2001-015-DP0344) and the

SRC Molecular Therapy Research Center in Sungkyunkwan

University.

References and Notes

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BM049563J

Figure 10. Transfection efficiency in 293 and HepG2 cells. Numbersin parentheses mean N/P ratios.

Figure 11. Serum effect on transfection efficiency of the polymer.Numbers in parentheses mean N/P ratios.


tae-il kim et al- pamam-peg-pamam: novel triblock copolymer as a biocompatible and efficient gene...

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