Preparation of pH-sensitive CaP nanoparticles coated with a phosphate-based block copolymer for efficient gene delivery

Article abstract of DOI:10.1016/j.polymer.2012.08.043

We have synthesized a phosphate-based block copolymer, PEG-b-PMOEP (poly(ethylene glycol)-b-poly(2-methacryloyloxyethyl phosphate)), with a narrow molecular weight distribution (PD = 1.06) by atomic transfer radical polymerization (ATRP), and have constructed calcium phosphate nanoparticles (CaPNs) coated with the block copolymer as an efficient and safe intracellular gene delivery carrier. The phosphate-mimic PMOEP block could be incorporated into the calcium phosphate (CaP) core to entrap pDNA, with the PEG block forming a shell to prevent uncontrolled growth of CaP precipitates and aggregates in physiological fluids. The CaPNs showed high colloidal stability at pH 7.4, but released entrapped pDNA at an endosomal pH of 5.0 through a pH-dependent protonation of phosphate moieties for efficient endosomal escape. The PEG-b-PMOEP/CaP/pDNA nanoparticles, which were formed simply by mixing, exhibited great potential as gene delivery carriers for future gene therapy applications due to their high transfection efficiency, low toxicity, and good stability under physiological conditions.

Full text of DOI:10.1016/j.polymer.2012.08.043

Polymer 53 (2012) 4678e4685
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Polymer
journal homepage: www.elsevier.com/locate/polymer
Preparation of pH-sensitive CaP nanoparticles coated with a phosphate-based
block copolymer for efficient gene delivery
Sangmok Jang a, Seonju Lee a, Heejin Kim a, Jiyeon Ham a, Ji-Hun Seo b, Yeongbong Mok a, Minwoo Noh a,
,
Yan Lee a
*
a Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151 747, Republic of Korea
b Department of Organic Materials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a phosphate-based block copolymer, PEG-b-PMOEP (poly(ethylene glycol)-b-
poly(2-methacryloyloxyethyl phosphate)), with a narrow molecular weight distribution (PD ¼ 1.06) by
atomic transfer radical polymerization (ATRP), and have constructed calcium phosphate nanoparticles
(CaPNs) coated with the block copolymer as an efficient and safe intracellular gene delivery carrier. The
phosphate-mimic PMOEP block could be incorporated into the calcium phosphate (CaP) core to entrap
pDNA, with the PEG block forming a shell to prevent uncontrolled growth of CaP precipitates and
aggregates in physiological fluids. The CaPNs showed high colloidal stability at pH 7.4, but released
entrapped pDNA at an endosomal pH of 5.0 through a pH-dependent protonation of phosphate moieties
for efficient endosomal escape. The PEG-b-PMOEP/CaP/pDNA nanoparticles, which were formed simply
by mixing, exhibited great potential as gene delivery carriers for future gene therapy applications due to
their high transfection efficiency, low toxicity, and good stability under physiological conditions.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 June 2012
Received in revised form
14 August 2012
Accepted 18 August 2012
Available online 24 August 2012
Keywords:
Block copolymer
Calcium phosphate
pH-sensitivity
1. Introduction
methods for applications in human gene therapy are still unsatis-
factory, only a small number of clinical trials have shown limited
Calcium phosphate (CaP), the major component of bone, has
been used as an important biomaterial for dental remodeling,
tissue engineering, and drug delivery [1e3]. Due to the strong
Coulombic interaction between calcium and phosphate ions that
leads to hydration resistance, CaP has low aqueous solubility, an
important characteristic for many of its applications. The addition
of DNA to calcium and phosphate ions while forming CaP precipi-
tates in aqueous solution allows the DNA to be incorporated as a co-
precipitate. The CaPeDNA co-precipitate can be readily internalized
into cells, and the entrapped DNA released into the cytosol,
a phenomenon that has been recognized as an effective method for
intracellular gene delivery since the 1960s [4,5].
As the concept of gene therapy through delivery of nucleic acid
drugs into cells has evolved as potential cures of human diseases,
various non-viral gene delivery methods have been developed
including polymereDNA complexes (polyplex) [6,7], lipideDNA
complex (lipoplex) [8,9], and electroporation [10,11] due to the
potential dangers of viral gene delivery methods [12,13]. Because
the efficiency and safety of developed non-viral gene delivery
success [14]. Therefore, research into the development of efficient
and safe gene delivery carriers is on-going, and the CaP co-
precipitation method has been revived as an important and reli-
able delivery device candidate due to its efficiency, biocompati-
bility, bioresorbability, and simple preparation. However, the
uncontrolled growth and rapid decrease of physiological stability of
CaP precipitates significantly limits their application as gene
delivery carriers in biological systems. Large aggregates over
several micrometers in diameter cannot be easily internalized into
cells via the endocytic process [15], and can also block the blood
stream in fine capillaries [16,17]. Additionally, the accumulation of
large aggregates in organs such as the lungs, skin, or intestines can
significantly reduce delivery efficiency and specificity [16].
In order to control the growth and to improve the physiological
stability of CaP precipitates with reproducible physicochemical
characteristics that provide gene delivery with greater efficiency
and safety, lipids and polymers have been used as detergents and
coating materials. However, the process of preparing CaP micro- or
nanoparticle emulsions in the presence of lipids [18] or polymers
[19] requires relatively complex steps that include sonication or
homogenization, which can damage DNA due to shearing forces.
Alternatively, CaP nanoparticles can be efficiently prepared by the
simple addition of a block copolymer during the co-precipitation
* Corresponding author. Tel.: þ82 2 880 4344; fax: þ82 2 871 2496.
E-mail address: gacn@snu.ac.kr (Y. Lee).
0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2012.08.043

S. Jang et al. / Polymer 53 (2012) 4678e4685
4679
process. If a block copolymer with a neutral hydrophilic block (e.g.
poly(ethylene glycol) (PEG)) and a negatively charged block (e.g.
poly(aspartic acid) (PAA)) is added to the calcium phosphate solu-
tion, the negatively charged block can be incorporated into the core
of the CaP precipitate through interactions between the calcium
ions and negatively charged moieties, and the neutral hydrophilic
block can form a shell to prevent uncontrolled aggregation through
steric repulsion [20e23].
In this study, we synthesized PEG-b-PMOEP (poly(ethylene
glycol)-b-poly(2-methacryloyloxyethyl phosphate)) with a narrow
molecular weight distribution (PD ¼ 1.06) by atomic transfer
radical polymerization (ATRP), then prepared pDNA-encapsulated
CaP nanoparticles (CaPNs) by coating with the PEG-b-PMOEP, and
measured the enhancement of gene delivery efficiency with the
nanocarrier. The PMOEP block, which resembles the head group of
phosphatidic acid (PA), has many phosphomonoester moieties that
strongly interact with calcium ions forming the CaP core, and the
PEG block with a sufficient length (Mn ¼ 5000) forms a hydrophilic
shell that can control the size of the CaP particle to approximately
200 nm (Fig. 1). Phosphomonoester moieties have two distinct pKa
values, pKa1 ¼ w3 and pKa1 ¼ w7 [24] unlike carboxylate moieties
with pKa of w4. It was expected that doubly charged phospho-
monoester moieties could stabilize CaP nanoparticles at neutral pH
via strong interaction with calcium ions, whereas the CaP nano-
particles could be destabilized by gradual protonation of the
phosphomonoester moieties during the acidification in the early
endosome allowing release of encapsulated nucleic acids. The
enhancement of gene delivery efficiency by the phosphate-based
polymer coating was analyzed by comparison with non-coated
CaP precipitates and other gene delivery methods.
2. Experimental
2.1. Materials
Poly(ethylene glycol) mono-methyl ether (PEG-OH; Mn ¼ 5000,
Mw ¼ 5300), 2-bromoisobutyryl bromide, triethylamine (TEA), 2-
hydroxyethyl methacrylate (HEMA), dimethyl chlorophosphate
(DCP), copper (I) bromide (CuBr), 2,20-bipyridine (Bpy), trime-
thylsilyl bromide (TMSBr), calcium chloride (CaCl2), 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
Fig. 1. Preparation of pDNA loaded PEG-b-PMOEP/CaP nanoparticles and its proposed release process to the endosome after endocytosis for gene delivery.

4680
S. Jang et al. / Polymer 53 (2012) 4678e4685
were purchased from SigmaeAldrich (St. Louis, MO, USA). Methanol
(MeOH), chloroform, and methylene chloride (MC) were purchased
from Daejung (South Korea). The luciferase assay system and
reporter lysis buffer were purchased from Promega (Madison, WI,
USA). Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin-EDTA,
phosphoesters [25]. After stirring at 0 ꢁC for 20 h, the solution was
poured into excess ether to precipitate the product. After drying the
product under vacuum, it was dissolved in MC (5 mL), and the
solution was dialyzed against MeOH, and then against deionized
water (M.W.C.O. ¼ 6000e8000). The final product (6) was obtained
by lyophilization (yield > 95%). From the 1H NMR spectrum
(Fig. 3(b)), the degree of polymerization (DP) of PMOEP units was
calculated to be 50. The molecular weight distribution of PEG113-b-
PMOEP50 was determined by gel permeation chromatography
(GPC) using a SuperdexÔ 75 column (GE healthcare, USA) cali-
brated by PEG standards. 50 mM TriseHCl (pH 7.4) was used as the
eluent at a flow rate of 0.7 mL/min at 35 ꢁC.
100
ꢀ
antibioticeantimycotic (penicillin, streptomycin, and
amphotericin B), and fetal bovine serum (FBS) were purchased from
GIBCO (Gaithersburg, MD, USA). Phosphate-buffered saline (PBS)
was purchased from Cambrex Bio Science (Walkersville, MD, USA).
All chemicals were used without further purification. The Micro
BCAÔ Protein Assay Kit was purchased from Pierce (Rockford, IL,
USA). A firefly luciferase expression plasmid, pCN-Luci, was con-
structed by subcloning the cDNA of Photinus pyralis luciferase with
the 21 amino acid nuclear localization signal of SV40 large T antigen
into pCN plasmids.
2.6. PEG-b-PMOEP/CaP/pDNA nanoparticle preparation
ExGen 500 was purchased from Fermentas (Burlington, Ontario,
Canada).
A solution of 2.5 M CaCl2 was added to a solution of pCN-Luci
DNA in distilled water to prepare a 2 ꢀ stock solution (Ca2þ
250 mM, pCN-Luci DNA 10 mg/mL). An aliquot of the stock solution
2.2. Synthesis of PEG macroinitiator (3)
was quickly added to an equal volume of 2 ꢀ PEG-b-PMOEP/
phosphate solution (pH 7.1, 50 mM HEPES, 6.0 mM Na2HPO4,
300 mM NaCl). After vigorous mixing for a few seconds, the
mixture was incubated at 47 ꢁC for 24 h. The size distribution and
zeta potential of nanoparticles in the suspension was analyzed at
37 ꢁC by dynamic light scattering using a Zetasizer 3500 (Malvern
Instruments, USA) equipped with a HeeNe ion laser at a wave-
length of 633 nm.
PEG-OH (1) (2.0 g, 0.40 mmol) and TEA (390
dissolved in 14 mL of MC. After cooling the solution with an ice
bath, 2-bromoisobutyryl bromide (2) (500 L, 4.0 mmol) was
slowly added to the vigorously stirred solution. The solution was
further stirred at room temperature for 24 h. The resulting PEG
macroinitiator (3) could be purified by precipitation in diethyl ether
(yield > 90%). The MALDI-TOF MS (Bruker Daltonics, Bremen,
Germany) spectra of 1 and 3 are shown in Figure S1.
mL, 2.8 mmol) were
m
2.7. Measurement of pCN-Luci DNA entrapment efficiency
2.3. Synthesis of MPDME (2-methacryloyloxyethyl
phosphoryldimethylester) monomer (4)
After the formation of PEG-b-PMOEP/CaP/pDNA nanoparticles,
the sample suspension was centrifuged at 15,000 ꢀ g for 30 min to
precipitate the nanoparticles. The pCN-Luci DNA concentration in
the supernatant was calculated from the absorbance at 260 nm.
Entrapment efficiency (%EE) was determined using the following
equation:
HEMA (4.85 mL, 40.0 mmol) and pyridine (3.22 mL, 40.0 mmol)
were dissolved in 15 mL of chloroform. DCP (15.7 mL, 120 mmol)
was added dropwise to the ice-cold solution under stirring. After
stirring at room temperature for 48 h, the reaction mixture was
diluted with chloroform (35 mL). The solution was then extracted
with 100 mL of 0.1 N HCl, and the organic layer was collected, dried
over MgSO4, and concentrated by rotary evaporation to yield
MPDME (4).
½Aꢂ0 ꢃ ½Aꢂs
%EE ¼
ꢀ 100
½Aꢂ0
where [A]0 and [A]s are the OD values of the initial pDNA solution
and the supernatant, respectively.
1H NMR (300 MHz, CDCl3,
3.80e4.00 (6H, e(CH2)2OPO(OCH3)2),
(CH2)2OPOe), 5.90e6.10 (2H, CH2]CCH3CO2e).
d
ppm):
d1.93 (3H, CH2]CCH3CO2e),
d
d4.20e4.50 (4H, eCO2
2.8. pH-Sensitive release of pCN-Luci DNA
d
The PEG-b-PMOEP/CaP/pDNA nanoparticle solution (PEG-b-
2.4. Synthesis of PEG-b-PMPDME (PEG-block-poly(2-
PMOEP concentration ¼ 500 g/mL) was centrifuged at 13,000 ꢀ g
m
methacryloyloxyethyl phosphoryldimethylester)) (5)
for 10 min. The supernatant was carefully removed, and the
precipitate was resuspended in two aqueous buffer solutions (pH
7.4 and pH 5.0). Each sample was dialyzed against buffer solution at
37 ꢁC using a D-Tube dialyzer (M.W.C.O. ¼ 6000e8000). Agarose
gel electrophoresis on 1.0% (w/v) agarose gel was used for analysis
of DNA release. After electrophoresis, the gel was stained with
PEG113-b-PMPDME50 (5) was synthesized by ATRP using 3 as
a PEG113-Br macroinitiator. 3 (0.20 g, 0.039 mmol) was added to
a solution of MPDME (460 mL, 2.0 mmol) in MeOH (4 mL). After
three freezeepumpethaw cycles, dried CuBr (12 mg, 0.078 mmol)
and bpy (24 mg, 0.16 mmol) were added to the solution under an
argon atmosphere. Polymerization was carried out at 40 ꢁC for 12 h.
After polymerization was complete, the solution was poured into
excess ether to precipitate the product. 5 was obtained by drying
the precipitate under vacuum (yield > 90%). The polymerization
efficiency from 3 was almost 99%. The 1H NMR spectrum of 5 is
shown in Fig. 3(a). From the 1H NMR spectrum, the degree of
polymerization (DP) of PMPDME units was calculated to be 50.
ethidium bromide solution (0.5
mg/mL), and the DNA band was
visualized on a UV illuminator. The intensity of the DNA band was
analyzed by a gel image analysis software (GelQuant.NET software,
Biochemlabsolutions.com).
2.9. Cytotoxicity measurement
To determine the cytotoxicity of the nanoparticles, MTT assays
were performed. HeLa cells were seeded in 96-well tissue culture
2.5. Synthesis of PEG-b-PMOEP (PEG-block-poly(2-
methacryloyloxyethyl phosphate)) (6)
dishes at 5000 cells/well in 90
24 h before transfection. After replacing the medium with fresh
complete medium, 30 L of each sample was added and the cells
were further incubated for 24 h. 20 L of filtered MTT solution
mL of DMEM containing 10% FBS for
PEG113-b-PMPDME50 (5) (190 mg) was added to a solution of
m
TMSBr (290
m
L, 2.2 mmol) in MC (10 mL) for deprotection of the
m

S. Jang et al. / Polymer 53 (2012) 4678e4685
4681
(2 mg/mL in PBS) was added to each well. After incubation for 4 h at
37 ꢁC, the medium was removed from the wells and 150
L of DMSO
m
was added to dissolve the formazan crystals. Absorbance was
measured at 570 nm using a microplate reader (Molecular Devices
Co., Menlo Park, CA, USA) and cell viability was calculated by
comparison with untreated control cells. Triplicate experiments
were performed for determining the average value and standard
deviation.
2.10. Cell culture and transfection
HeLa cells (human cervical cancer cells) were grown in complete
medium (DMEM supplemented with 10% FBS and 1% antibiotics) at
37 ꢁC in a humidified 5% CO2 incubator. HeLa cells were seeded at
a density of 15,000 cells/well into 24-well tissue culture dishes in
600 mL of complete medium for 24 h before transfection. 120 mL of
all samples (pDNA only, CaP normal, CaPNs, ExGen 500) with
complete medium (DMEM supplemented with 10% FBS and 1%
antibiotics) were added to the cells at a final pCN-Luci DNA
concentration of 0.5e4 mg/mL and the cells were further incubated
for 48 h.
For the transfection with ExGen 500, N/P ratio of 6 was used as
an optimal transfection condition. For the luciferase assay, the
growth medium was removed and the cells were washed with PBS
Fig. 2. Synthetic scheme of PEG-b-PMOEP (6) by ATRP.
and lysed for 30 min at room temperature with 150 mL of reporter
lysis buffer. Luciferase activity in the transfected cells was
measured using an LB 9507 luminometer (Berthold, Germany) with
both 1H NMR and GPC (Table 1). From 1H NMR, the degree of
polymerization (DP) of the PMOEP block was calculated to be 50.
The polydispersity (PD) of 6 was 1.06, which represents a successful
ATRP of the phosphate-based block copolymer with a narrow
molecular weight distribution.
10
mL of the lysate dispensed into a luminometer tube and the
automatic injection of 50
mL of Luciferase Assay Reagent. The
protein concentration of the lysate was determined using a MicroÔ
BCA protein assay kit. Luciferase activity was measured in terms of
relative light units (RLU) and the final values were determined as
RLU/mg of total protein in the lysate. Triplicate experiments were
performed for determining the average value and standard devia-
tion. A t-test was performed between individual treatment groups,
using the software Graphpad Prism 5.04 (GraphPad software, Inc.).
3.2. Preparation of PEG-b-PMOEP/CaP/pDNA nanoparticles
The newly synthesized phosphate-based block copolymer, PEG-
b-PMOEP, was used to prepare pDNA-encapsulated CaPNs for gene
delivery. When calcium ions were mixed with phosphate ions,
mixed salts including monocalcium phosphate (Ca(H2PO4)2) and
dicalcium phosphate (CaHPO4) were formed depending on the pH
of the solution. DNA could be readily incorporated into the CaP
precipitates during the precipitation process. Growth of the
precipitates was difficult to be controlled and very heterogeneous
precipitates were formed without stabilizing reagents. In presence
of PEG-b-PMOEP, the phosphate-based PMOEP block could be
incorporated into the CaP precipitate core, and the neutral hydro-
philic PEG block could coat the surface of the CaP. Because a PEG of
short length is not sufficient for complete shielding and steric
stabilization of nanoparticles to prevent proteinenanoparticle
interactions in physiological fluids [29], we chose the PEG block
with Mn ¼ 5000 for stabilization of CaP particles.
3. Results and discussion
3.1. Synthesis of PEG-b-PMOEP
ATRP is one of the most powerful methods to control the
polymerization of a wide range of monomers [26]. In this study,
a diblock copolymer with a neutral hydrophilic PEG block and
a negatively charged phosphate block, PEG-b-PMOEP (6), was
synthesized by ATRP (Fig. 2) to form a surface coating for CaP
nanoparticles (CaPNs). The PEG macroinitiator, PEG-Br (3), was
synthesized from PEG-OH (1) and 2-bromoisobutyryl bromide (2).
Because it was difficult to determine the conjugation efficiency by
1H NMR due to the relatively small number of terminal residues,
MALDI-TOF was used as an alternative method to compare the
molecular weight difference between 1 and 3. Figure S1 shows that
the conversion from 1 to 3 was almost 100% efficient. The phos-
phoryldimethylester monomer, MPDME (4), was prepared from
HEMA and DCP since ATRP is often hampered by the presence of
acid groups [27,28]. In addition, MPDME (4) was quite soluble in
alcoholic ATRP solvents that were used to dissolve the PEG
macroinitiator.
pDNA-encapsulated CaP nanoparticles were simply prepared by
mixing Ca2þ/pDNA and phosphate/PEG-b-PMOEP solutions. The
colloidal stability of CaPNs was highly dependent upon the
concentration of the phosphate-based block copolymer. CaPNs less
than 200 nm in diameter formed only with concentrations of the
block copolymer over 380
mg/mL (Fig. 4(a)). In the range of 400e
500 g/mL, PEG-b-PMOEP/CaP/pDNA nanoparticles of approxi-
m
mately 180 nm in size were obtained. These CaPNs showed
a unimodal size distribution with polydispersity indices (PDI)
ranging from 0.1 to 0.2, which was confirmed by dynamic light
scattering (DLS) analysis (Fig. 4(bec)). The incubation temperature
was another important factor for successful formation of PEG-b-
PMOEP/CaP/pDNA nanoparticles. CaPNs with narrower size distri-
butions were generally obtained as the temperature was increased
(data not shown). Considering both narrow size distributions and
PEG-b-PMPDME (5) was synthesized from the PEG macro-
initiator (3) using CuBr and bpy as the catalyst and ligand, respec-
tively (Fig. 3(a)). PEG-b-PMOEP (6) was easily obtained through
demethylation of 5. After demethylation, proton peaks at d3.80e
4.00 ppm corresponding to dimethyl esters of 5 had completely
disappeared from the 1H NMR spectrum (Fig. 3(b)). The molecular
weight distribution of the product block polymer was analyzed by

4682
S. Jang et al. / Polymer 53 (2012) 4678e4685
Fig. 3. 1H NMR spectra of (a) PEG-b-PMPDME (5) and (b) PEG-b-PMOEP (6) in CD3OD.
thermal denaturation of pDNA at higher temperatures, we deter-
mined the optimal temperature to be 47 ꢁC for the transfection
experiments shown below.
Table 1
Measurement of molecular weights of polymers.
PD (Mw/Mn)b
a
b
b
Mn1
Mn2
Mw
5.15 ꢀ 103
4.95 ꢀ 103
5.10 ꢀ 103
1.03
1.06
3.3. Measurement of entrapment efficiency and zeta potential
PEG-Br initiator (3)
PEG-b-PMOEP (6)
1.56 ꢀ 104
1.72 ꢀ 104
1.82 ꢀ 104
The values are determined from 1H NMR spectra.
The values are determined by GPC.
a
The entrapment efficiency of pDNA in the polymer-coated
CaPNs was measured by UV absorbance at 260 nm. Fig. 5 shows
b

S. Jang et al. / Polymer 53 (2012) 4678e4685
4683
Fig. 5. Entrapment efficiency (C) and zeta potential (:) of pDNA loaded PEG-b-
PMOEP/CaP nanoparticles (PEG-b-PMOEP concentration of CaP normal, CaPN 2, CaPN
3 ¼ 0, 440, 500 g/mL, respectively). Error bars indicate the standard deviation (n ¼ 3).
m
CaP particles were strongly dependent upon the polymer coating.
CaP normal showed a zeta potential of about þ33 mV, but polymer-
coated CaPNs showed a zeta potential closed to zero, which sug-
gested that neutral hydrophilic PEG block formed a shell on the
surface of CaPNs.
3.4. pH-Sensitive release of DNA from PEG-b-PMOEP/CaP/pDNA
nanoparticles
Because the electrostatic interaction between Ca2þ and H2PO4ꢃ is
weaker than between Ca2þ and HPO24ꢃ, the aqueous solubility of
CaP increases as the pH decreases [30]. It was also expected that the
interaction between PEG-b-PMOEP and Ca2þ would decrease as
a result of gradual protonation of phosphate groups in the PMOEP
backbone.
Nanoparticles of several hundred nanometers in size are readily
internalized into cells through the endocytic process, an important
pathway for efficient intracellular drug/gene delivery [31]. Subse-
quently, internalized nanoparticles should escape the endosome
into the cytosol before full acidification (pH 4) or maturation to
a lysosome in which degrading enzymes are activated [32]. It was
expected that the dissolution of CaP into calcium and phosphate
ions at acidic pH can increase osmotic pressure in the endosome to
induce endosomal membrane rupture by osmotic swelling and
released pDNA from CaP can escape the endosome efficiently [33].
Therefore, release of pDNA from CaPNs at the endosomal pH 5.0
and normal physiological pH 7.4 were compared. Fig. 6 shows the
result of agarose gel electrophoresis after incubation of PEG-b-
PMOEP/CaP/pDNA nanoparticles at the two different pH values. At
pH 7.4, no release of pDNA from the nanoparticles was detected
even after 24 h and the nanoparticles maintained a narrow size
distribution with high colloidal stability even after 72 h (data not
shown). On the other hand, pDNA release began after 1 h at pH 5.0
with about 70% of the pDNA released from the CaPNs after 1 h and
almost 95% released after 2 h as measured by a gel image analysis
software. These results strongly support the supposition that PEG-
b-PMOEP/CaP/pDNA nanoparticles can dissociate and the released
pDNA can escape the endosome in the weakly acidic early endo-
somal state before degradation and inactivation of pDNA in the
lysosome.
Fig. 4. Formation of PEG-b-PMOEP coated CaP nanoparticles with pDNA entrapment.
(a) Polymer concentration dependence of the size of the CaP nanoparticles. (b) Polymer
concentration dependence of the PDI of the CaP nanoparticles. (c) The sized distri-
bution of the CaP nanoparticles (PEG-b-PMOEP concentration ¼ 500
mg/mL). All data
points are corresponding to CaP nanoparticles prepared at 47 ꢁC.
the entrapment efficiency and zeta potential for three different
PEG-b-PMOEP concentrations (0, 440, and 500 g/mL). All samples
had showed that more than 80% of the pDNA loaded into the CaP
particles, which suggests that the pDNA incorporation efficiency
was not closely related with the polymer coating but was affected
by the co-precipitation process. However, the surface charges of
m
3.5. Cytotoxicity measurement of PEG-b-PMOEP and PEG-b-
PMOEP/CaP/pDNA nanoparticles
Cytotoxicity of free PEG-b-PMOEP and CaPNs was measured
separately by the MTT assay. Compared to ExGen 500 (linear

4684
S. Jang et al. / Polymer 53 (2012) 4678e4685
was also confirmed until the pDNA concentration of 4
mg/mL
(Fig. 7(b)). The cytotoxicity results support the potential of PEG-b-
PMOEP/CaP/pDNA nanoparticles as safe gene delivery carriers for
use in biosystems.
3.6. Transfection of HeLa cells by PEG-b-PMOEP/CaP/pDNA
nanoparticles
Transfection experiments of HeLa cells were performed with
PEG-b-PMOEP/CaP/pDNA nanoparticles. As shown in Fig. 8(a), the
PEG-b-PMOEP-coated CaPNs showed about 2-fold higher tranfec-
tion efficiency than normal CaP precipitates. By controlling the size
of CaP precipitates to approximately 200 nm, the transfection
efficiency could be significantly enhanced, probably due to more
effective endocytic uptake of CaPNs compared to simple hetero-
geneous CaP precipitates with an average size over several
micrometers [19]. As shown in confocal laser scanning microscopy
(CLSM) images (Figure S2), normal CaP precipitates were difficult to
be internalized into HeLa cells in spite of their positive surface
charges, the PEG-b-PMOEP-coated CaPNs with almost neutral
surface charges were efficiently internalized by endocytic
pathways.
Fig. 6. pH-sensitive release of pDNA from PEG-b-PMOEP/CaP nanoparticles according
to the incubation time on pH 7.4 and pH 5.0, respectively (PEG-b-PMOEP
concentration ¼ 500
mg/mL).
polyethylenimine), a commercially available transfection reagent,
which showed about 20% of cell viability at the concentration of
10
mg/mL, free PEG-b-PMOEP showed almost no cytotoxicity even
at the polymer concentration of 40
mg/mL (Fig. 7(a)). Additionally,
non-toxicity of PEG-b-PMOEP/CaP/pDNA nanoparticles (CaPN 2)
Fig. 8. (a) Transfection efficiency of PEG-b-PMOEP/CaP nanoparticles on HeLa cells at
a pDNA concentration of 1.39
mg/mL (PEG-b-PMOEP concentration of CaPN 1, CaPN 2,
Fig. 7. (a) Cytotoxicity of PEG-b-PMOEP (B) and ExGen 500 (C) at various polymer
concentration on HeLa cells. (b) Cytotoxicity of CaPN 2 (B) and ExGen 500/DNA
polyplex (N/P ¼ 6) (C) at various pDNA concentration on HeLa cells. Error bars
indicate the standard deviation (n ¼ 3).
CaPN 3 ¼ 380, 440, and 500
mg/mL, respectively). (b) Transfection efficiency of PEG-b-
PMOEP/CaP nanoparticles (CaPN 2) (B) and ExGen 500 (N/P ¼ 6) (C) at various pDNA
concentration on HeLa cells. Statistical significance was represented as p value. Error
bars indicate the standard deviation (n ¼ 3).

S. Jang et al. / Polymer 53 (2012) 4678e4685
4685
The effect of the initial PEG-b-PMOEP concentrations on trans-
Appendix A. Supplementary material
fection efficiency was negligible. For comparison with other non-
viral gene delivery carriers, ExGen 500 was used as a positive
control. Our CaPNs showed higher transfection efficiency compared
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.polymer.
2012.08.043.
to ExGen 500 at the pDNA concentration range of 0.5e4
mg/mL
(Fig. 8(b)). The transfection efficiency of CaPNs and ExGen 500
polyplexes was gradually increased as the pDNA concentration was
increased. It is probably due to increased uptake of CaPNs at higher
pDNA concentrations.
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This work was supported by the Basic Science Research Program
(2011-0002301) through the National Research Foundation (NRF)
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