Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Bone-specific poly(ethylene sodium phosphate)-bearing

biodegradable nanoparticles
Yuya Hirano a , Yasuhiko Iwasaki b,c,∗
a
Graduate School of Science and Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-8680, Japan
b
Department of Chemistry and Materials, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka,
564-8680, Japan
c
ORDIST, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-0836, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Chemotherapy is the most reliable treatment for osteoporosis and osseous metastases. To facilitate better
Received 24 September 2016 drug delivery for bone treatments, a novel preparation of polymeric nanoparticles with high affinity to
Received in revised form 10 January 2017 bone has been prepared. Two-step synthesis of cholesteryl-functionalized poly(ethylene sodium phos-
Accepted 13 February 2017
phate) (Ch-PEPn ·Na) was performed via ring-opening polymerization of cyclic phosphoesters and the
Available online 16 February 2017
demethylation. The molecular weight of Ch-PEPn ·Na could be well controlled by changing the ratio of
cholesterol and cyclic phosphoesters. Because Ch-PEPn ·Na exhibits an amphiphilic nature in aqueous
Keywords:
media, Ch-PEPn ·Na-bearing nanoparticles (PEPn ·Na NPs) were prepared by a solvent evaporation tech-
Polyphosphoester
Nanoparticle nique. The size of the nanoparticles investigated in the current study is approximately 100 nm, which
Drug delivery system was determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Due to
Bone therapy the presence of highly water-soluble polymer chains, dispersion of PEPn ·Na NPs in aqueous media was
Ring-opening polymerization stable for at least 1 week. Hemolytic activity of PEPn ·Na NPs was found to be low and PEPn ·Na NPs did not
Biodegradable polymer disintegrate mammalian cell membranes. Additionally, cytotoxicity of PEPn ·Na NPs was not observed at
concentrations below 100 ␮g/mL. The adsorption of PEPn ·Na NPs on hydroxyapatite (HAp) microparticles
was studied in comparison with poly(ethylene glycol) nanoparticles (PEG NPs). Both PEPn ·Na NPs and
PEG NPs adsorbed well onto HAp microparticles in distilled water with binding equilibrium constants
(KHAp ) for PEPn ·Na NPs and PEG NPs of 3.6 × 106 and 7.9 × 106 , respectively. In contrast, only PEPn ·Na NPs
adsorbed onto HAp microparticles in a saline phosphate buffer. Moreover, the adsorption of PEPn ·Na NPs
onto HAp microparticles occurred even in the presence of 1.2 mM calcium ions or low-pH media. The
affinity of the nanoparticles to bovine bone slices was also studied, with the result that large quantities
of adsorbed PEPn ·Na NPs were observed on the slices by scanning electron microscope.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction for metastatic cancer. Present chemotherapy procedures for bone

Bone is the most common metastatic site in epithelial cancers
such as breast, prostate, and lung cancers [1,2]. Bone metastasis
often causes symptoms, such as pathological fractures, hypercal-
cemia, and spinal cord compression, which significantly decrease
the quality of life (QOL) of patients [3,4]. Treatments for bone
metastatic cancers currently involve performing a combination of
surgery, radiation therapy, and chemotherapy. Chemotherapy in
particular is believed to be one of the most essential treatments
∗ Corresponding author at: Department of Chemistry and Materials, Faculty of
Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho,
Suita-shi, Osaka, 564-8680, Japan.
E-mail address: yasu.bmt@kansai-u.ac.jp (Y. Iwasaki).
metastasis use anti-cancer drugs as well as bone-modifying agents,
such as bisphosphonates (BPs) [5–9] and denosumab [10,11], in
clinical practice. Both these agents can inhibit or prolong the start-
up period of skeletal-related events, such as surgery and radiation
therapy, which greatly contribute to improving the QOL of patients
[12,13]. Bone-modifying agents are also applied to improve the
delivery of anticancer drugs to metastatic sites in bone. A variety
of BP-bearing drug carriers have been proposed for this purpose
because their ability to form bidentate or tridentate chelates with
calcium ions results in a high affinity to hydroxyapatite (HAp),
the inorganic component of bone [14–19]. For example, BPs have
been immobilized to bind to the side chains of hydrophilic poly-
mers [16], liposomes [17], and polysaccharides such as pullulan
[18]. However, BPs do not have sufficient functionality to conjugate
biofunctional molecules or anti-cancer drugs. Moreover, BP-related

http://dx.doi.org/10.1016/j.colsurfb.2017.02.015
0927-7765/© 2017 Elsevier B.V. All rights reserved.
 Y. Hirano, Y. Iwasaki / Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110
osteonecrosis of the jawbone has been reported [20,21]. To achieve
therapeutic bone treatments safely and efficiently, new drug deliv-
ery materials and systems are required.
Here, we propose poly(ethylene sodium phosphate) (PEPn ·Na)
as a new polymeric candidate material with an affinity to bone
substrates. PEPn ·Na is an analog of polyphosphoesters and has a
phosphodiester backbone [22]. Polyphosphoesters have recently
been focused as new biodegradable polymers that have mimetic
structures of nucleic acids [23–29]. Compared with conven-
tional aliphatic polyesters, the molecular functionalization of
polyphosphoesters is more easily achieved because various cyclic
phosphoesters, which function as monomers, can be obtained by a
simple condensation reaction between an alcohol and 2-chloro-2-
oxo-1,3,2-dioxaphosphorane (COP) [30,31]. That is, theoretically,
any alcohol can be introduced into polyphosphoesters. Further-
more, the solubility of polyphosphoesters can be controlled by the
substituent side chain groups. We have successfully polymerized
cyclic phosphoester monomers using organocatalysts [28], which
have advantages of being able to undergo ring-opening polymer-
ization in living manner and to generate polyphosphoesters with
a narrow molecular weight distribution (Mw /Mn < 1.1). Addition-
ally, end-functionalized polyphosphoesters are easily synthesized
because alcohol molecules can be used as polymerization initiators.
As described above, cyclic monomers for polyphosphoesters can
be obtained by a simple condensation reaction and various func-
tional groups can be introduced into the side chains of polymers,
allowing versatile control of polyphosphoester solubility. The bio-
compatibility of polyphosphoesters has also been determined in
previously published works, and several biomedical applications
have been proposed such as drug- [24,25,27,32] and gene delivery
[33,34] and tissue engineering [35,36].
Polyphosphoester ionomer, consisting of phosphotriester and
phosphodiester units, is one of the water-soluble polyphospho-
esters we have studied previously [37]. Interestingly, the amount
of polyphosphoester ionomer adsorption onto HAp substrates
increased with more phosphodiester units (ionized units) in the
polyphosphoesters [38]. Moreover, polyphosphoester ionomers
clearly exhibited superior physicochemical properties for mineral-
ization and biocompatibility when compared with BPs. According
to the earlier experiments, fully ionized PEPn ·Na is expected to
show high affinity to HAp. A more recent study evaluated the
effects of PEPn ·Na on bone cell viability and activity [22], and found
that PEPn ·Na exhibited excellent biocompatibility with osteoblasts.
In contrast, selective inhibition of osteoclast adhesion and func-
tion was observed following cultivation with PEPn ·Na. PEPn ·Na
should thus be considered as a unique polymer with diverse uses
for bone treatment; however, no studies have yet addressed the
use of PEPn ·Na for processing drug carriers. In the current study,
amphiphilic PEPn ·Na was newly synthesized to prepare biodegrad-
able polymer nanoparticles that can interact strongly with bone.
The characterization, biocompatibility, and mineral affinity of the
nanoparticles are also described.
2. Materials and methods
2.1. Materials
2-Chloro-2-oxo-1,3,2-dioxaphospholane (COP) was kindly sup-
plied by NOF Corporation (Tokyo, Japan) and cholesterol
was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).
1,8-Diazabicyclo[5,4,0]-undec-7-ene (DBU) was obtained from
Sigma-Aldrich (Saint Louis, USA), and was purified by dis-
tillation under reduced pressure, in which a fraction of bp
130 ◦ C/3.0 mmHg (lit.: bp 80 ◦ C/0.6 mmHg) was used. 2-Methoxy-
2-oxo-1,3,2-dioxaphospholane (MP) was synthesized using a
105
previously described method [22,37]. Other chemical reagents
were purchased from Wako Pure Chemical Inc. (Tokyo, Japan) and
were used without further purification.
2.2. Synthesis of cholesteryl-terminated poly(ethylene phosphate)
Scheme 1 shows the synthesis route of cholesteryl-terminated
poly(ethylene phosphate) (Ch-PEPn ·Na; n: number of repeating
units), an amphiphilic polyphosphoester. 2.76 g (20 mmol) MP was
placed into a sufficiently dry 20 mL Schlenk tube equipped with
a three-way stopcock followed by drying under reduced pressure
for 2 h. A certain amount of cholesterol, which had previously been
dried under reduced pressure overnight, was dissolved in 2 mL of
dichloromethane. The cholesterol/dichloromethane solution and
DBU (1.0 equivalent to cholesterol) were added under an Ar gas
atmosphere. The mixture was stirred with a magnetic stirrer in
an ice bath for 1 h and was further stirred at room temperature
for 5 h. The reaction was quenched by the addition of acetic acid
(1.0 equivalent to DBU). The synthesized choresteryl-terminated
poly(MP) (Ch-PMPn ; n: number of repeating units) was purified
by reprecipitation from toluene followed by diethyl ether, placed
into a desiccator, and dried under reduced pressure. The Ch-PMPn
was dissolved in 10 mL of distilled water and mixed with a 30%
trimethylamine aqueous solution (7.88 g; equivalent to 2.0 MP
units). The mixture was stirred at room temperature for 24 h and
subsequently stirred with 22.2 g of a cationic ion exchange resin
®
(Amberllite IR-120, Merck KGaA, Darmstadt, Germany) at room
temperature for 1 h. In order to completely transform polymers
containing quaternized amimonium salts into polyacids (polyethy-
lene phosphate), this procedure was repeated twice. The polymer
solution was then dialyzed against distilled water for 6 h and freeze-
dried in order to concentrate the solution. After concentrating, the
pH of the aqueous solution was adjusted to 7.0 using a sodium
hydroxide aqueous solution and the polymer solution was again
dialyzed against distilled water. Finally, Ch-PEPn ·Na was obtained
in the solid state.
The apparent critical micelle concentration (cmc) value for Ch-
PEPn ·Na in aqueous media was determined using a previously
described method using pyrene as a fluorescence probe [39].
2.3. Preparation of Ch-PEPn ·Na-bearing poly(L-lactic acid) (PLLA)
nanoparticles
The Ch-PEPn ·Na-bearing nanoparticles (PEPn ·Na NPs) were pre-
pared using a solvent evaporation method [40–43]. Briefly, a 40 mL
aqueous solution containing 10 mg/mL Ch-PEPn ·Na was placed in
a sample tube and stirred at 450 rpm while cooled in an ice bath.
1 mg PLLA was dissolved in 1.0 mL chloroform and then added drop-
wise into the aqueous polymer solution. The mixture was sonicated
using a probe-type generator (Sonifier 250, Branson, CT, USA) for
30 min and kept under reduced pressure for 30 min to evaporate the
chloroform. The nanoparticles formed were fractionated by cen-
trifugation at 45000 rpm at 4 ◦ C for 1.5 h (himac 70MX, Hitachi Koki
Co., Ltd., Tokyo, Japan). The nanoparticles were resuspended as a
precipitate in distilled water and centrifuged again under the same
conditions. This procedure was repeated three times to completely
remove any free Ch-PEPn ·Na.
Poly(ethylene glycol)-bearing nanoparticles (PEG NPs) were
also prepared by using cholesteryl-terminated PEG (Mw = 6000;
NOF Corporation, Tokyo, Japan) as an emulsifier. The preparation
process of PEG NPs was identical to that of the PEPn ·Na NPs.
The particle size, intensity size distribution, and ␨-potential of
the nanoparticles were determined using a Malvern Instruments
Zetasizer (Worcestershire, UK). Scattering was performed with a
vertically polarized incident beam at a wavelength of 633 nm sup-
plied by a He-Ne ion laser with a scattering angle of 90◦ . The
106 Y. Hirano, Y. Iwasaki / Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110
Scheme 1. Synthesis route for amphiphilic poly(ethylene phosphate).
morphology of the nanoparticles was observed by transmission
electron microscopy (TEM; JEM-1400, JEOL, Tokyo, Japan).
2.4. Cytotoxicity assay
Mouse osteoblastic cells (MC3T3-E1) were used for cell culture
experiments because they are a well-characterized osteoblast-like
cell line that can serve as a model for endogenous osteoblasts
[44,45]. The cells were maintained in a culture medium [␣-media
modified essential medium (␣-MEM); Thermo Fisher Scientific,
Inc., Grand Island, NY, USA] containing 10% fetal bovine serum
and 1% antibiotic-antimycotic. The cells were routinely cultured in
tissue-culture polystyrene flasks (Asahi Glass Co., Ltd, Tokyo, Japan)
at 37 ◦ C in a humidified atmosphere of air containing 5% CO2 . After
treatment with trypsin- ethylenediaminetetraacetic acid (EDTA;
Gibco, Invitrogen Corporation, USA), the MC3T3-E1 osteoblasts
were harvested.
The suspension of MC3T3-E1 cells (100 ␮L; 5 × 104 cell/mL) in ␣-
MEM was introduced into a 96-well tissue-culture plate (Thermo
Scientific, Rochester, USA) and incubated in a culture medium at
37 ◦ C with 5% CO2 . After 24 h of cultivation, Dulbecco’s phosphate-
buffered saline (PBS (−)) (10 ␮L) containing either PEP106 ·Na NPs or
PEG NPs was added to each of the wells and the cells were cultivated
for a further 24 h. To determine the cell viability, a Cell Counting Kit-
8 (CCK-8; DOJINDO LABOLATORIES, Kumamoto, Japan) was used.
2.5. Hemolysis test
Hemolysis testing was carried out according to a previously
described method [46]. Blood samples (20 mL) were collected from
healthy volunteers with a 30 mL syringe, to which was added 3 mL
of a 3.8 wt% trisodium citrate aqueous solution. The collected blood
samples were centrifuged for 15 min at 1200 rpm (AllegraTM 21R
Centrifuge, Beckman Coulter, Brea, CA, USA) in order to precipitate
the erythrocytes. The supernatant and interface were discarded
and Hank’s balanced salt solution (HBSS) in the amount of 1.5
times that of the precipitated erythrocytes was added and resus-
pended. The solution was then centrifuged for 10 min at 2800 rpm
and the supernatant and interface were discarded followed by dilu-
tion with HBSS. This suspension was then diluted 10 times with
HBSS. 200 ␮L of PEP106 ·Na NPs suspended in HBSS and 800 ␮L of
the erythrocytes/HBSS suspension were mixed followed by incu-
bation for 3 h at 37◦ (Dry Block Bath EB-603, AS-ONE, Osaka, Japan).
The final preparation of PEP106 ·Na NPs had a concentration in the
range of 1 × 10−4 –1 × 10−1 mg/mL. After the mixture was stored
for 3 h at 37 ◦ C, it was centrifuged for 10 min at 2800 rpm and the
absorbance of the supernatant at 405 nm was measured using a
microplate reader (Model 680 Microplate reader, Bio-Rad, Her-
cules, CA, USA). Solutions of pure HBSS and Triton-X 100/HBSS
(1 wt%) were used as positive and negative controls, respectively,
in place of the PEP106 ·Na NPs suspension and were mixed with the
erythrocytes/HBSS suspension.
2.6. Binding affinity of PEP106 ·Na NPs for HAp surfaces
For fluorescent labeling of nanoparticles, 0.2 mg of oxonol VI
was dissolved with PLLA in chloroform when the nanoparticles
were prepared. The nanoparticles were suspended in various
aqueous media such as distilled water, PBS(−), PBS containing
1.2 mM calcium ions (PBS(+)), and 50 mM Tris-buffer (pH = 5.7).
®
HAp microparticles (10 mg, 22.82 m2 /g; Macro-Prep ; Ceramic
Hydroxyapatite Type II 80 ␮m, Bio-Rad, Hercules, CA, USA) were
added to the suspension and incubated for 3 h with gentle mix-
ing. The supernatant was then collected and further centrifuged at
6200 rpm for 10 s (PMC-060, TOMY SEIKO Co., Ltd., Tokyo, Japan)
to precipitate nanoparticles adsorbed on the HAp. The centrifuged
supernatant (100 ␮L) was mixed with ethanol and chloroform
(1:2:1 vol ratio) to dissolve the nanoparticles. The fluorescence
intensity (ex 570 nm, em 628 nm), which is due to eluted oxonol
VI from the dissolved nanoparticles, was recorded with a fluo-
rescence spectrophotometer (F-2500, Hitachi, Tokyo, Japan). The
amount of nanoparticles adsorbed on the HAp microparticles was
then determined by comparing the concentration of nanoparticles
in the supernatant with that in the original nanoparticle solution.
The amount of adsorbed nanoparticles was calculated relative to
the unit surface area of the HAp microparticles.
2.7. Adsorption of PEP106 ·Na NPs on bone slices
Bone slices were prepared as previously described [47]. Briefly, a
frozen bovine cortical femur was cut with a diamond saw (Buehler,
Lake Bluff, IL, USA) into slices of 130–180 ␮m thickness. The bone
slices (5 mm × 5 mm) were rinsed by ultrasonication in distilled
water, then soaked in an aqueous suspension containing 0.8 mg/mL
of nanoparticles and stored for 3 h with gentle shaking. The spec-
imens were then rinsed with distilled water three times and
freeze-dried under vacuum pressure. Subsequently, the specimen
surfaces were coated with Au-Pd by a MSP-S1 magnetron sputter
(Vacuum Device Inc., Ibaraki, Japan) and observed with a scanning
electron microscope (SEM; JSM-6700, JEOL, Tokyo, Japan).
3. Results and discussion
3.1. Synthesis and characterization of Ch-PEPn ·Na
Scheme 1 and Supporting Table S1 illustrate the synthesis route
and characterization of Ch-PEPn ·Na, respectively. The number of
repeating units (n) in the polymers and the molecular weight of
Ch-PEPn ·Na were determined by proton nuclear magnetic reso-
nance (1 H NMR; ␣-500, JEOL, Tokyo, Japan) end group analysis. The
ring opening polymerization of cyclic phosphoesters proceeds in a
controlled/living fashion with organocatalysts [28]. The molecular
weight of Ch-PEPn ·Na was then controlled by changing the fractions
of MP and cholesterol. During the transformation of polytriphos-
phoester (PMPn ) into polydiphosphoester (PEPn ), no change in the
number of repeating units (n) in the polymers was observed (See
supporting Table S1).
Ch-PEPn ·Na consists of a hydrophilic chain and a hydropho-
bic cholesteryl end. Therefore, Ch-PEPn ·Na exhibits an amphiphilic
nature and form micelles in aqueous solution. In order to deter-
mine the cmc of Ch-PEPn ·Na in a Tris-HCl buffer solution (10 mM,
pH = 7.4), pyrene was used as a fluorescence probe. The ratios of
the pyrene peak intensities at 383 and 373 nm (I383 /I373 ) were
plotted as a function of Ch-PEPn ·Na concentration because the flu-
orescence emission spectra of pyrene are polarity dependent [39].
In the process of aggregation, the I383 /I373 values of pyrene are
 Y. Hirano, Y. Iwasaki / Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110
Fig. 1. TEM images of PEP106 ·Na NPs (a) and PEG NPs (b). Scale bar = 200 nm.
Fig. 2. Particle size and PDI of PEP106 ·Na NPs (a) and PEG NPs (b) suspended in water (䊉) and PBS(−) (䊏) during days 1–7. (PEP106 ·Na NPs; n = 3, PEG NPs; n = 3).
lower at concentrations below the cmc value, whereas the ratios
are enhanced at higher Ch-PEPn ·Na concentrations when pyrene
is encapsulated in the micelles. Therefore, the two fitting lines
reflecting the two states intersect at the turning point, the horizon-
tal ordinate of which determines the cmc values to be 1.0 × 10−2
(Ch-PEP24 ·Na), 2.0 × 10−2 (Ch-PEP46 ·Na), and 4.0 × 10−2 g/dL (Ch-
PEP106 ·Na), as shown in Supporting Table S1 and Supporting Fig.
S1. The cmc increased with an increase in the molecular weight
of the polymer. The phosphodiester polymer chain of Ch-PEPn ·Na
is highly water soluble polymers [37]. Hydrophobic association
of cholesteryl groups can be then weakened by long hydrophilic
polyphosphoesters.
3.2. Preparation of PEPn ·Na NPs
The preparation of nanoparticles was performed by solvent
evaporation technique with amphiphilic polymer concentration
above the cmc. DLS data for PEPn ·Na NPs and PEG NPs are shown
in Table 1 and Supporting Fig. S2. All nanoparticles prepared in
this study had diameters of approximately 100 nm (polydisper-
sity index (PDI), 0.1–0.3). The surface ␨-potential of PEPn ·Na NPs
was around −70 mV and was highly negatively charged, while that
of PEG NPs was around −40 mV. Fig. 1 shows the TEM images of
PEPn ·Na106 NPs and PEG NPs, indicating that the NPs are spherical
in shape and that the diameters coincide well with the DLS data.
To evaluate the stability of PEP106 ·Na NPs in the dispersion
medium, PEP106 ·Na NPs and PEG NPs were suspended in distilled
107
water and PBS(−) at a concentration of 0.5 mg/mL and the parti-
cle size and PDI were measured every day by DLS for 7 days. As
shown in Fig. 2, both PEP106 ·Na NPs and PEG NPs suspended in
water showed high colloidal stability over one week. The negatively
charged ␨-potential of nanoparticles in water contributes to their
high dispersion ability. When PEP106 ·Na NPs and PEG NPs were
suspended in PBS(−), the ␨-potential values of the nanoparticles
decreased and the ␨-potential of PEG NPs became neutral due to
the charge screening effect by the ionic environment (Table 2), indi-
cating that the dispersion ability of PEG NPs in PBS(−) was inferior
to that of PEP106 ·Na NPs. The negative charge of PEP106 ·Na NPs in
PBS(−) resulted in excellent dispersion stability.
The solvent evaporation technique for preparation of nanopar-
ticles composed of hydrophobic inner core and an outer shell of
hydrophilic polymers is reliable method to make nanoparticles.
This procedure has been used for preparation of various nanopar-
ticulate drug carriers incorporating hydrophobic molecules [48].
3.3. Biocompatibility of PEP106 ·Na NPs
Fig. 3 shows the viability of MC3T3-E1 cells in contact with vari-
ous concentrations of PEP106 ·Na NPs dissolved in PBS(−). MC3T3-E1
cells in contact with PEP106 ·Na NPs displayed high viability values
similar to those with PEG NPs, and the 50% inhibitory concentration
(IC50 ) value of PEP106 ·Na NPs was not confirmed in this concentra-
tion range.
108 Y. Hirano, Y. Iwasaki / Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110

Table 1
Characterization of PEPn ·Na NPs and PEG NPs.

Sample SizeTEM (d nm)a SizeDLS (d nm)b PDIb ␨-potential(mV)b

PEP24 ·Na NPs – 99.2 0.24 −72.7

PEP46 ·Na NPs – 77.5 0.24 −64.8
PEP106 ·Na NPs 88.2 108.0 0.14 −67.2
PEG NPs 94.0 119.8 0.15 −39.2
a
Calculated from TEM images using SemAfore 5.21 (JEOL, Tokyo, Japan).
b
Determined DLS measurement in distilled water.

Table 2
Binding constant (KHAp ) of nanoparticles to HAp.

Nanoparticle Medium pH KHAp ( × 106 M−1 ) R2 ␨-potential (mV)

PEP106 ·Na NPs Water – 3.55 ± 2.02 0.99 −67.2

PBS(−) 7.4 9.75 ± 1.03 0.98 −30.9
PBS(+) 7.4 10.08 ± 1.45 0.99 −34.6
Tris-buffer 5.7 7.91 ± 3.43 0.90 −49.2
PEG NPs Water – 7.93 0.87 −39.2
PBS(−) 7.4 N.D. – −3.5

HBSS for a concentration range of 0.1–100 ␮g/mL, indicating that

there was no hemolysis activity. It was therefore concluded that
PEP106 ·Na NPs do not damage cell membranes.

3.4. Binding affinity of PEP106 ·Na NPs on HAp surfaces

Various tetracyclines, bisphosphonates, acidic oligopeptides,

Fig. 3. Viability of MC3T3-E1 cells in contact with nanoparticles (n = 3).
chelating compounds, and salivary proteins have all been employed
to target bone diseases. These compounds bind to the inorganic
HAp portion of bone and have specificity for a certain HAp crystal
size [50]. Recently, we reported that polyphosphoester ionomers
had high affinity for HAp surfaces, or the inorganic component
of bone [38]. Because of their high affinity to HAp surfaces, it is
therefore expected that PEP106 ·Na NPs can induce extravasation
from blood vessels by enhanced permeability and retention effects
and bind strongly to bone surfaces. In order to determine their
binding affinity for HAp surfaces, PEP106 ·Na NPs were placed in
contact with HAp microparticles in various media, such as distilled
water, PBS(−), 1.2 mM of calcium ions containing PBS (PBS(+)), and
Tris-buffer (50 mM, pH = 5.7). According to preferred encapsula-
tion of hydrophobic molecules into nanoparticles, oxonol VI-loaded
PEP106 ·Na NPs were used to quantify the amount of PEP106 ·Na NPs
adsorbed onto the HAp surface. Supporting Fig. S3 shows the bind-
ing isotherms of nanoparticles on HAp surfaces. When distilled
water was used as a medium, the amount of adsorbed PEP106 ·Na
NPs on the HAp surface is similar to that of PEG NPs. In contrast,
the amount of adsorbed PEP106 ·Na NPs to HAp in PBS(−) is much
larger than that of PEG NPs.
In order to quantify the binding affinity on HAp surfaces, the
binding equilibrium constant for HAp (KHAp ) was calculated from
the Langmuir equation as follows:

qm · KHAp · c
q= (1)
1 + KHAp · c

Fig. 4. Hemolytic activity of nanoparticles (n = 3).
In vivo hemolysis is a serious phenomenon, in which the ery-
throcyte membrane is destroyed by certain factors, such as viruses,
chemicals, and low osmotic pressure, and hemoglobin is released
from the erythrocyte [49]. In vivo hemolysis results in anemia
and the released hemoglobin causes serious complications such as
renal failure, stroke, and heart attack. To investigate the effect of
PEP106 ·Na NPs on erythrocyte membranes, the PEP106 ·Na suspen-
sion and the erythrocyte suspension were mixed. The hemolysis
rates of PEP106 ·Na NPs, shown in Fig. 4, were similar to that of
where q is the quantity of adsorbed nanoparticles per unit amount
of HAp (␮g/mg), qm is the maximum quantity of adsorbed nanopar-
ticles (␮g/mg), KHAp is the binding equilibrium constant (M−1 ), and
c is the nanoparticle concentration (mg/mL). To calculate KHAp , we
utilized the coverage value (coverage:  = q/qm ) such that the Lang-
muir equation can be transformed as follows:
1 1
=1+ (2)
 KHAp · c
The coverage was calculated using the density of PLLA, and the
Avogadro number (NA ) was utilized to express KHAp as M−1 [51].
 Y. Hirano, Y. Iwasaki / Colloids and Surfaces B: Biointerfaces 153 (2017) 104–110 109

Fig. 5. SEM images of bovine bone in contact with 0.8 mg/mL of PEP106 ·Na NPs and PEG NPs suspended in PBS(−). Scale bar = 1 ␮m.

Table 2 shows the KHAp values for PEP106 ·Na NPs and PEG NPs hemolysis activity or cytotoxicity against MC3T3-E1 cells. PEPn ·Na
to HAp. PEP106 ·Na NPs suspended in PBS(−) exhibited a high KHAp NPs also showed high in vitro affinity to bone components, includ-
value, while the KHAp value for PEG NPs could not be obtained. ing hydroxyapatite (HAp) and bone slices. Additionally, the affinity
Moreover, we evaluated the binding affinity of PEP106 ·Na NPs of PEPn ·Na NPs to HAp was not disrupted by the presence of cal-
on HAp in the presence of calcium ions [38]. It was predicted cium ions or low-pH conditions, which promote bone resorption by
that 1.2 mM of free calcium ions would inhibit the adsorption of activated osteoclasts. PEPn ·Na NPs therefore have high potential for
PEP106 ·Na NPs onto HAp surfaces due to possible electrostatic inter- delivery of hydrophobic drugs to bone.
action between PEP106 ·Na and calcium ions. However, the KHAp
value of PEP106 ·Na NPs suspended in PBS(+) was equally as high as Acknowledgments
that in PBS(−), indicating that free calcium ions do not influence the
adsorption of PEP106 ·Na NPs onto HAp surfaces. It is assumed that This work was supported by JSPS KAKENHI Grant Number
the interaction of calcium ions with the PBS(+) and PEP106 ·Na NPs (26107719, 15H03520, 15H03526, and 26107719), and also by
can be easily exchanged with other ions. However, the exchange Hitachi Metals Materials Science Foundation 2014.
of interaction between PEP106 ·Na NPs and HAp surface does not
easily occur because both PEP106 ·Na NPs and HAp surfaces contain Appendix A. Supplementary data
many interaction sites. The adsorption of PEP106 ·Na NPs onto HAp
surfaces would therefore not be suppressed even in the presence Supplementary data associated with this article can be found, in
of various other ions found in body fluids. the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.02.
Osteoclasts play an important role in bone metastasis by cre- 015.
ating space for cancer cells via bone resorption [52]. Osteoclasts
degrade the bone mineral component by secreting protons through
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