PDEA-coated magnetic nanoparticles for gene delivery to Hep G2 cells

Biotechnology and Bioprocess Engineering 18: 648-654 (2013)

DOI 10.1007/s12257-012-0720-z

PDEA-coated Magnetic Nanoparticles for Gene Delivery to Hep G2

Cells

Hanwen Sun, Xinjun Zhu, Lianying Zhang, Xiangling Gu, Jinghe Wang, Jing Li, and Yancong Zhang

Received: 4 November 2012 / Revised: 2 February 2013 / Accepted: 7 February 2013

© The Korean Society for Biotechnology and Bioengineering and Springer 2013

Abstract Poly(2-(diethylamino)ethyl methacrylate) coated

magnetic nanoparticles (PDEA-MNPs) were synthesized

as a new gene nanocarrier to delivery plasmids (pEGFP-

N1 and pRL-TK) into human hepatoma (Hep G2) cells.

The PDEA-MNPs shows the pH-sensitive property. These

nanoparticles are positively charged at acidic pH and

negatively charged at neutral or alkaline pH. The PDEA-

MNPs exhibited a low cytotoxicity in Hep G2 cells.

PDEA-MNPs could bind and protect DNA from DNase I

degradation. The transfection study demonstrated that the

PDEA-MNPs could carry plasmid into Hep G2 cells and

exhibited a high gene transfection efficiency. These results

indicated that the novel magnetic nanoparticles could

enhance gene transfection in vitro and hold the potential to

be a promising non-viral nanodevice.

Keywords: PDEA, magnetic nanoparticles, pH-sensitive,

gene delivery

1. Introduction

Successful gene therapy challenge relies heavily on the

fabrication of suitable gene carriers that can efficiently

deliver specific genes to the desired cells with minimum

cytotoxicity [1,2]. Both viral and nonviral vectors have

been used for gene delivery, viral vector systems have

generally been identified as being useful, owing to their

superior ability to deliver and express genes to target cells.

However, the use of viral vectors for gene delivery can be

associated with severe inflammation and immunological

problems [3,4]. In order to overcome theses problems,

nonviral vector systems have been designed to avoid the

aforementioned problems, while retaining their functional

properties [5,6].

In recent years, magnetic micro- or nanoparticles have

emerged as promising new platforms in biomedical applica-

tions, such as protein and enzyme immobilization, magnetic

resonance imaging (MRI), target delivery of drugs, magne-

tofection, etc [7-10]. In order to absorb negative charged

DNA on their surface, magnetic nanoparticles are usually

modified with polyethylenimine (PEI) or other cationic

polymers [11,12]. Although PEI evidences a high potential

for transfection efficiency, the associated cytotoxicity

remains a problem [13].

Poly(2-(dimethylamino)ethyl methacrylate) (PDEA) is a

water-soluble polymer which is sensitive to both temperature

and pH changes, it is being extensively studied for numerous

potential applications, particularly in the biomedical field,

such as drug delivery systems, bioseparations, biosensors,

and tissue engineering [14-16]. dical field, such as drug/

gene or transfection efficiency, the cytotoxicity remained

an unsolved In our previous works, we have described the

synthesis of polymer coated magnetic nanoparticles via

photochemical polymerization [17-19]. In this paper, we

synthesized poly(2-(dimethylamino)ethyl methacrylate)-

coated magnetic nanoparticles (PDEA-MNPs) and investi-

gated the properties of plasmid DNA adsorption, cytotoxicity

of the polymer coated magnetic nanoparticles, and their

efficiency of application to gene delivery in human hepatoma

(Hep G2) cells.

Hanwen Sun, Xinjun Zhu, Lianying Zhang, Xiangling Gu, Jinghe Wang,Jing Li, Yancong ZhangDezhou Institute of Advanced Materials, Dezhou University, Dezhou 253-023, China

Hanwen Sun*

Key Laboratory of Functional Polymer Materials, Ministry of Education,Institute of Polymer Chemistry, Nankai University, Tianjin 300-071, ChinaTel: +86-534-898-5972; Fax: +86-534-898-9506E-mail: [email protected]

RESEARCH PAPER

PDEA-coated Magnetic Nanoparticles for Gene Delivery to Hep G2 Cells 649

2. Materials and Methods

2.1. Materials

2-(dimethylamino)ethyl methacrylate (DEA), 3-(4,5)-

dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide

(MTT) and Polyethylene glycol diacrylate (PEGDA,

Mn~550) were purchased from Sigma-Aldrich; Plasmid

EGFP-N1 and RL-TK were purchased from Invitrogen

(Eugene, OR, USA) and Clontech companies, respectively.

RPMI-1640 medium and fetal bovine serum (FBS) were

purchased from Invitrogen Corporation (Carlsbad, CA,

USA). The transfection reagent TansFastTM was purchased

from Promega Co., (Madison, WI, USA).

2.2. Preparation and characterization the PDEA-MNPs

Superparamagnetic Fe3O4 nanoparticles were prepared by

a partial reduction co-precipitation method which was

described in detail in a previous report [18]. PDEA-MNPs

were prepared in an aqueous system as described previously

[17]. Briefly, 430 µL of DEA, 40 µL of PEGDA and

10 mg of Fe3O4 nanoparticles were mixed in 60 mL water

in a quartz cell. The mixture was mechanically stirred for

30 min to maximize the surface adsorption of DEA and

PEGDA on the Fe3O4 nanoparticles. Afterwards, the

mixture was irradiated by two 8-W UV lamps for 20 min.

Nitrogen gas was bubbled throughout the reaction process.

The PDEA-MNPs were separated from the reaction

mixture by a magnet and were washed several times with

distilled water. At last the PDEA-MNPs were dissolved in

distilled water and stored in a freezer until use.

2.3. Characterization

FT-IR spectra of Fe3O4 nanoparticles and PDEA-MNPs

were analyzed with a Nicolet Avatar 370 Fourier transform

infrared spectroscopy (FT-IR) in a wave number range of

4000 ~ 500/cm. Morphology of PDEA-MNPs was observed

with transmission electron microscopy (TEM, Hitachi 600).

The size and zeta-potential of PDEA-MNPs were detected

by photo correlation spectroscopy (PCS, Zetasizer Nano

ZS, Malvern Instruments Ltd.). The samples for FT-IR and

TEM were vacuum dried at room temperature and the

sample for PCS were diluted to 10-5 mol/L in water.

2.4. Cell viability assay

The cytotoxicity of PEDA-MNPs was assessed through

MTT assay. The Hep G2 cells were seeded in 96-well

plates at 2 × 104 cells per well and were cultured in RPMI-

1640 supplemented with 10% FBS, 100 U/mL penicillin,

and 100 mg/mL streptomycin for 20 h at 37oC under a 5%

CO2 atmosphere, and were then exposed to different

concentrations of PDEA-MNPs. After 2, 12, or 24 h

incubation, the PEDA-MNPs were removed, and the cells

were washed twice with PBS. Then 200 µL of fresh

medium and 20 µL of MTT solution (5 mg/mL in PBS)

were added into the well. The cells were cultured for

another 4 h at 37oC. The solution was aspirated gently and

150 µL of DMSO was added to dissolve the formazan

crystals. After shaking for 10 min, the absorbance of each

well at 490 nm was measured by Thermo Scientific

Varioskan Flash (Winooski, VT, USA). The cell viability

assay was performed in triplicate, and the results were

expressed as the mean percentage of cell viability relative

to untreated cells.

2.5. Combination analysis PDEA-MNPs and plasmid

DNA

The DNA/PDEA-MNPs complexes were prepared and

determined as follows: 5 µg of pRL-TK DNA in 20 µL

PBS buffer (pH 5.0) was mixed with different amounts of

PDEA-MNPs (5 ~ 120 µg). After shaking for 10 min at

room temperature, 5 µL of the incubated DNA/ PDEA-

MNPs complexes was mixed with 1 µL of 6 × loading

buffer, and was loaded into a 1% agarose gel containing

ethidium bromide (0.5 µg/mL). The electrophoresis experi-

ment was carried out with 1×TAE buffer at a constant

voltage of 80 V. The pDNA bands were then visualized

under a UV transilluminator at a wavelength of 365 nm.

2.6. DNA release from DNA/PDEA-MNPs complexes

To study the DNA release behavior in vitro, 1.0 mg of

PEDA-MNPs with 50 µg DNA in the form of sediment

were transferred to a clean 5 mL centrifugal tube with 3 mL

PBS (pH 7.2 and 5.0, respectively). The sealed tube was

placed in a water bath maintaining temperature at 37oC. At

specified collection times, 100 mL of sample was taken

from the tube and DNA concentration was measured. The

samples in the 5 mL tube were replenished with 100 mL

fresh PBS at 37oC. Triplicate samples were analyzed.

The total released DNA Mi at time i was calculated from

Eq. (1)

Mi = CiV + ΣCi-1 Vs (1)

Where Ci is the concentration of DNA in the elution

buffer at time i, V is the total volume of elution buffer

(3 mL) and Vs is the sample volume (0.1 mL).

2.7. DNase I protection assay

The DNase I protection assay was carried out according to

the previous method [20]. Briefly, 5 µg of pDNA was

incubated with PDEA-MNPs at room temperature to form

DNA/PDEA-MNPs complexes. The complexes were treated

with DNase I (1 U/µg of DNA) at 37oC for 10 min,

followed by heat denaturation of DNase I at 70oC for

10 min. Then 6 µL of EDTA (0.25 M, pH 8.0) and 12 µL

650 Biotechnology and Bioprocess Engineering 18: 648-654 (2013)

of SDS (15%) were added and the samples were incubated

at 37oC for 1 ~ 2 h, resulting in pDNA dissociation from

complexes. The pDNA released from the complexes were

visualized by 1% agarose gel electrophoresis as described

above.

2.8. Transfection the PDEA-MNPs-pDNA to Hep G2

cells

Hep G2 cells were seeded in 96-well plates at 2 × 104 cells

per well and were then cultured in RPMI-1640 supplemented

for 20 h at 37oC under a 5% CO2 atmosphere prior to

transfection. Plasmid (0.2 µg) mixed with different amount

of DNA-PDEA-MNPs at 50 µL of PBS buffer (ph 5.0) and

incubated at room temperature for 20 min for complexes

formation. Then the mixture was dissolved in 200 µL of

serum-free RPMI-1640 and was added into each well.

After 2 h of incubation at 37oC in a 5% CO2 atmosphere,

the transfection solutions were aspirated and substituted by

complete culture medium. After additional 24 h incubation,

the cells were directly observed by Zeiss inverted microscope

(Werk, Gottingen, Germany).

The pRL-TK plasmid was further used to evaluate the

transfection efficiency. The transfection experiment was

performed as above. After 24 h incubation, the medium

was removed and cells were washed twice by cold PBS

(pH 7.4), the cells were then lysed using 20 µL of Promega

Passive lysis buffer. The cell lysate was transferred into

100 µL Stop & GloTM for luminescence measurements.

The relative light units (RLUs) were measured with a

Thermo Scientific Varioskan Flash (Winooski, VT, USA).

3. Result and Discussion

3.1. Synthesis and characterization of PDEA-MNPs

Our previous studies demonstrated that the monomer DEA

and cross-linker PEGDA can be absorbed onto the surface

of Fe3O4 nanoparticles in the mixture. When irradiated

under UV light, the monomer DEA was polymerized with

the cross-linker PEGDA on the surface of the Fe3O4

nanoparticles. Then PDEA-MNPs were obtained by coating

the Fe3O4 nanoparticles with a cross-linked poly DEA shell.

Fig. 1 shows the FT-IT spectra of naked Fe3O4 and

PDEA-MNPs. For the naked Fe3O4, the peak at 576/cm

related to the Fe-O group. For the IR spectra of PDEA-

MNPs, the band of 1724/cm was assigned to the stretching

vibration of C=O and the peak 1103/cm to −C−O bending

vibration of the PDEA and PEGDA. The peak at 1246/cm

was correlated to the C−N stretching vibrations. Furthermore,

a sharp peak 576/cm appeared in the PDEA-MNPs spectra.

The results indicated that the PDEA-MNPs were synthesized

successfully via photochemical polymerization.

PEGDA is widely used in bio- and tissue engineering,

due to its biocompatibility, ease of photo-polymerization

and tailoring of mechanical properties [21]. In this study,

we used PEGDA as the cross-linker, which can improve

the biocompatibility of the PDEA-MNPs. The TEM image

showed that these nanoparticles have a spherical shape

(Fig. 2), and the average size in aqueous measured by

photon correlation spectroscopy (PCS) was 43 nm, with a

polydispersion index of 0.241 (Fig. 3).

Zeta potential measurements are a convenient way to

characterize the eletrostatic properties of vesicles, as the

zeta potential is a measurement of the electrical charge

close to the surface of the vesicles [22,23]. The zeta

potential of PDEA-MNPs was determined as a function of

pH. As can be seen from Fig. 4, the zeta potential varied

from +41.7 mV (pH 3.0) to −33.1 mV (pH 10), indicating

Fig. 1. FT-IR spectra of (A) naked Fe3O4 nanoparticles and (B)PDEA-MNPs.

Fig. 2. Transmission electron microscopy of the PDEA-MNPs.


that the sign of the surface charge of the PDEA-MNPs was

sensitive to the bulk pH, yielding positively charged nano-

particles at acidic pH and negatively charged nanoparticles

at neutral and alkaline pH.

3.2. Cytotoxicity of PDEA-MNPs

The cell toxicity of PDEA-MNPs instinct on target cells

was determined by the conventional MTT method, and the

results are shown in Fig. 5A. The PDEA-MNPs exhibited

a dose and time responsive effect on cell viability. No

significant reduction in viability was observed over a period

of 24 h with doses of up to 100 µg/mL, and the PDEA-

MNPs did not exhibit apparent toxicity to Hep G2 cells

after incubation for 12 h (200 µg/mL) or 2 h (250 µg/mL).

These results indicated that the PDEA-MNPs did not

possess a highly toxic nature.

In order to investigate the effect of PDEA modification

on the cell viability, the Hep G2 cells were incubated with

naked and PDEA-modified Fe3O4 nanoparticles for 12 h and

the cell viability was determined by MTT. We can see from

Fig. 5B that as compared to the naked Fe3O4 nanoparticles

the PDEA modification reduced cell toxicity.

3.3. Plasmid DNA bonds to PDEA-MNPs

Enhanced green fluorescent protein encoding DNA plasmid

(pEGFP-N1) and the Renilla luciferase encoding plasmid

pRL-TK were propagated in E. coli DH5-α and were

purified using the Plasmid Giga Kit (Qiagen, GmbH,

Germany) according to the manufacture’s instructions.

Purified plasmids were dissolved in 1×TE buffer and the

concentration was determined by Thermo Scientific Inc.

Nanodrop 1000 spectrophotometer (MA, USA). The purified

plasmid has an A260/A280 ratio of 1.86, indicating that

the plasmid has high purity, and was suitable for the

transfection study.

The PDEA-MNPs were positively charged at pH 5.0

(zate-potential: +40 mV, Fig. 4), instilling these particles

with the ability to bind negatively charged DNA molecules.

The behavior of DNA in agarose gel was blocked by the

huge PDEA-MNPs (compared with the nucleic acids

molecular). The agarose gel retardation assay indicated that

complete retardation of 5 µg plasmid DNA was achieved

at the nanoparticle weight of 80 µg, as shown in Fig. 6.

Fig. 3. Particle size of the PDEA-MNPs measured by PCS.

Fig. 4. Zeta-potential of the PDEA-MNPs vs. pH.

Fig. 5. Cell viability of (A) Hep G2 cells treated with different concentrations of PDEA-MNPs and (B) Hep G2 cells treated with PDEA-MNPs and naked Fe3O4 nanoparticles. *p < 0.05, **p < 0.01, and ***p < 0.001.


1 mg of PEDA-MNPs could bind about 60 µg of pDNA.

This binding capacity was equal to the polyethylenimine

(PEI) [24] or 3-[2-(2-aminoethylamino)-ethylamino]-

propyltrimethoxysilane (AEEA) [25] modified magnetic

nanoparticles.

Protection of plasmid DNA from nucleases is one of the

most important properties for efficient gene delivery in vitro

as well as in vivo. To test whether the PDEA-MNPs can

protect adsorbed plasmid DNA from nucleases digestion,

the nanoparticles were exposed to DNase I for 30 min.

Fig. 7 showed that naked pDNA was completely degraded

with no band observed, while at the same concentration of

DNase I, the pDNA extracted from complexes remained

intact. The results of DNase I protection assays show that

PDEA-MNPs are able to effectively protect plasmid DNA

from DNase I digestion, thus implying application prospects

in gene delivery.

3.4. DNA release from the DNA/PDEA-MNPs complexes

Fig. 8 shows the release behavior of DNA from DNA/

PDEA-MNPs complexes in PBS at pH 5.0 and 7.2 over a

time period of 5 h. It can be seen that the DNA release

curve showed a sustained release behavior and that the

DNA was progressively released by desorption and

diffusion to the PBS at pH 7.2, while there was no obvious

DNA release at pH 5.0. The difference in DNA release

behavior at different pH values can be attributed to the pH

sensitivity of the PDEA-MNPs. The PDEA-MNPs surface

has a positive charge at pH 5.0, which means that they

could tightly bind the negatively charged DNA. However,

the PDEA-MNPs surface was negatively charged at PBS

pH 7.2, which will make the DNA release slowly from the

PDEA-MNPs.

The pH-responsive property is very useful for delivering

genes to cells. The PDEA-MNPs can tightly bind and

protect DNA once the nanoparticles are internalized and

transferred to lysosomes. The pH within the lysosome is

about 4 ~ 5 [26]. In lysosomes, the PDEA-MNPs are

positively charged, and thus, can bind the DNA tightly.

Therefore, the DNA is not released into the lysosome.

When the nanoparticles escape into the cytosol (pH 7.2)

the DNA will be released from the complexes.

3.5. DNA transfection efficiency of DNA/ PDEA-MNPs

To determine whether the PDEA-MNPs can carry plasmid

into Hep G2 cells, plasmid EGFP-N1 was used as a model

Fig. 6. Agarose gel electrophoresis patterns of complexes. M:marker, line 1, 2, 3, 4, 5, and 6: 5, 10, 40, 80, 100, and 120 µg ofPDEA-MNPs combined with 5 ìg of pRL-TK DNA.

Fig. 7. Agarose gel electrophoresis of pDNA/PDEA-MNPscomplexes after being incubated with DNase I. Lines 1 ~ 5: 5 µgof pDNA incubated with 0, 5, 10, 40, and 80 µg of PDEA-MNPs.

Fig. 8. DNA release from DNA/PDEA-MNPs complexes.


gene in this study. The expression of EGFP gene was

detected by fluorescence microscopy. As can be seen from

Fig. 9, the efficiency of EGFP expression can be enhanced

significantly in the presence of MNPs compared with the

naked plasmid. It is clear that the PDEA-MNPs can absorb

plasmid DNA to mediate gene transfer in cultured Hep G2

cells.

To investigate the effect of mass ratio of nanoparticles/

DNA on the efficiency of gene delivery, the nanoparticle/

DNA complexes were added to the Hep G2 cells at

different mass ratios (0.2 µg DNA mixed with different

PDEA-MNPs), Transfection reagent TranfastTM was used

as a positive controls. The results illustrated that the mass

ratio of nanoparticle/DNA affects the gene delivery efficiency

(Fig. 10). The gene delivery efficiency was PDEA-MNPs

mass-dependent: The more PDEA-MNPs were added, the

more DNA can be bound on the nanoparticles and carried

into Hep G2 cells, and the highest gene delivery efficiency

was obtained at 50 µg. The gene delivery efficiency was

higher than that of Transfection reagent TranfastTM in this

experiment. This may attributed to the pH-sensitive polymer

on the MNPs releasing the plasmid efficiently, while

internalized in the cells. It illustrates that the most optimal

mass ratio of nanoparticles/DNA is 50 µg/0.2 µg for increased

performance in gene delivery experiments.

The successful delivery of pEGFP-N1 and pRL-TK gene

to Hep G2 cells demonstrated that the PDEA-MNPs can be

employed for gene vector to deliver gene to mammalian

cells. Thereby their potential as a gene carrier and may

play an important role in improving efficiency of gene

transfection and gene therapy.

4. Conclusion

In summary, the PDEA coated magnetic nanoparticles were

successfully synthesized via UV irradiation. The PDEA-

MNPs are spherical shape with the average diameter about

43 nm. The PDEA-MNPs were positively charged at acidic

pH and negatively charged at neutral or alkaline pH. MTT

study showed that these nanoparticles have low cytotoxicity

in Hep G2 cells. Agarose gel electrophoresis experiments

show that the PDEA-MNPs can bind and protected plasmid

against degradation of exonuclease. The transfection

efficiency of the PDEA-MNPs was determined to be as

powerful as commercial gene transfection reagent. These

nanoparticles may be promising for the delivery of nucleic

acid for in vitro and in vivo applications.

Acknowledgements

We are grateful to Shandong Provincial Natural Science

Foundation, China (No. 2009ZRA14029, No. ZR2010BL001),

Shandong Provincal Young and Middle Aged Scientists

Research Awards Foundation (No. BS2011CL001) and

Shandong Provincal Science and Technology Development

Project (No. 2011GGA14032, NO.2011YD02080) for

financial support.

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PDEA-coated magnetic nanoparticles for gene delivery to Hep G2 cells