Design, synthesis, and miniemulsion polymerization of new phosphonate surfmers and application studies of the resulting nanoparticles as model systems for biomimetic mineralization and cellular uptake

Article abstract of DOI:10.1002/chem.201103256

Heterophase polymerizations have gained increasing attention in the past decades, especially as the decoration and functionalization of the particle surface for further applications gets more and more into focus. One promising approach for the functionalization exclusively on the particle surface is the use of surfmers (surfactant and monomer). Herein, we present the synthesis of a new family of surfmers and their use for decorating nanoparticles with phosphonate groups through miniemulsion polymerization. Furthermore the synthesis of a dye-labeled functional surfmer provided an elegant manner to evaluate and get deeper insights about its copolymerization. Additionally, potential applications of the synthesized particles in biological studies as well as their use as template for biomimetic mineralization are presented.

Full text of DOI:10.1002/chem.201103256

FULL PAPER
DOI: 10.1002/chem.201103256
Design, Synthesis, and Miniemulsion Polymerization of New Phosphonate
Surfmers and Application Studies of the Resulting Nanoparticles as Model
Systems for Biomimetic Mineralization and Cellular Uptake
Rꢀdiger Sauer,[a] Pablo Froimowicz,*[a] Katrin Schçller,[a] Jens-M. Cramer,[a]
Sandra Ritz,[a] Volker Mailꢁnder,[a, b] and Katharina Landfester*[a]
Abstract: Heterophase polymerizations
have gained increasing attention in the
past decades, especially as the decora-
tion and functionalization of the parti-
cle surface for further applications gets
more and more into focus. One promis-
ing approach for the functionalization
exclusively on the particle surface is
the use of surfmers (surfactant and mo-
nomer). Herein, we present the synthe-
sis of a new family of surfmers and
their use for decorating nanoparticles
with phosphonate groups through mini-
emulsion polymerization. Furthermore
the synthesis of a dye-labeled function-
al surfmer provided an elegant manner
to evaluate and get deeper insights
about its copolymerization. Additional-
ly, potential applications of the synthe-
sized particles in biological studies as
well as their use as template for bio-
mimetic mineralization are presented.
Keywords: biomimetic mineraliza-
tion · cell uptake · miniemulsion
polymerization
surfmer
· nanoparticles ·
Introduction
able to synthesize well defined surface-functionalized nano-
particles without the need for any further chemical post-
functionalization and working under surfactant-free condi-
tions. The resulting latexes were subsequently demonstrated
to be highly applicable as a model system for different stud-
ies, as templates for biomimetic mineralization, and cell-
uptake experiments.
The synthesis of tailored surface-functionalized nanoparti-
cles is increasingly attracting the attention of researchers
due to the vast variety of applications in which they are ap-
plied in different fields,[1] such as generation and exploita-
tion of inorganic-organic hybrid nanomaterials through min-
eralization of organic templates[2] or coating the inorganic
moiety with organic layer(s);[3] in biological[4] and medical
researches[5] for cell uptake,[3a] or drug release,[6] and in poly-
mers and materials sciences for studying stimuli-responsive-
ness.[7] However, one of the major challenges in the genera-
tion of these surface-functionalized nanoparticles is how to
address an adequate organization of the matter to introduce
the desired functionalities at the required location already
during their preparation, thus avoiding post-polymerization
chemical modifications and/or unnecessary purification
steps. In this paper we show that with the use of new syn-
thetic surfmers (one class of reactive surfactants), we were
As aforementioned, among the different kinds of reactive
surfactants we decided to exploit the chemistry of surfmers
because they are amphiphilic compounds behaving simulta-
neously as a surfactant and a co-monomer. Etymologically,
the term “surfmer” is an acronym formed from the words
surfactant (surface-active agent) and monomer. Thus, as any
other surfactant, surfmers play a crucial role in heterophase
polymerizations such as emulsion and miniemulsion poly-
merizations. Although these two heterophase polymeri-
zation techniques have many similarities (including compart-
mentalization),[8] their particle nucleation mechanisms are
different. Whereas in emulsion polymerization it is micellar
nucleation that dominates,[9,10] in miniemulsion polymeri-
zation the particle nucleation occurs in the stabilized mono-
mer droplets.[8b,11,12] Thus, the role of the surfactant during
the polymerization process is different for each technique,
but once the polymerization is finished, surfactants are, in
both cases, responsible for keeping the synthesized nanopar-
ticles stable in the continuous phase. It is known that con-
ventional surfactants are physically adsorbed on the polymer
particles and may migrate inward or desorb from the prod-
uct causing their destabilization.[12,13] To avoid these negative
features of conventional surfactants, surfmers (among other
reactive surfactants) have attracted increasing atten-
tion.[14,15,16] These reactive surfactants participate in the poly-
[a] R. Sauer, Dr. P. Froimowicz, K. Schçller, J.-M. Cramer, Dr. S. Ritz,
Dr. V. Mailꢀnder, Prof. Dr. K. Landfester
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Fax : (+49)6131-379-370
E-mail: landfester@mpip-mainz.mpg.de
froimowicz@mpip-mainz.mpg.de
[b] Dr. V. Mailꢀnder
III. Medical Clinic (Hematology, Oncology, and Pulmology)
University Medicine of the Johannes-Gutenberg University Mainz
Langenbeckstr. 1, 55131 Mainz (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103256.
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merization process and become constituents of the polymer
backbone; therefore, they are not physically adsorbed but
covalently attached to the surfaces of the polymer parti-
cles,[12] making any migration nor desorption of the surfmers
no longer possible.
nate,[24,25] or phosphate,[24,25] functional groups. However, in
most cases, the functional groups were not used for any fur-
ther application.
Organophosphorous compounds have received increasing
interest in many different research areas and applications
fields, for example, biology,[5] biomedicine,[26] and technolo-
gy.[27] Although most of the organophosphorous-containing
polymerizable compounds known from literature, such as
acrylates or methacrylates, consist of short alkyl spacers be-
tween the two functionalities (therefore viewed as conven-
tional co-monomers), it has been established that for many
scientific and technological applications, long alkyl-chain
spacers are desirable.[25] These long-chain phosphorous-con-
taining compounds may be viewed as surfactants and even
as phospholipids analogues, and similar structures bearing
polymerizable groups may be considered as surfmers. In this
context, Francovꢂ and Kickelbick[24–25] described the synthet-
ic pathway of a family of methacrylate-functionalized phos-
phonate amphiphiles containing alkyl spacers with a variable
length, their micellization in aqueous media, and final poly-
merization to crosslink the nanospheres. The final product
showed that the generated nano-objects presented a large
size distribution. This inconvenience should be solved
straightforwardly by using miniemulsion polymerization as
an alternative for generating phosphonate-functionalized
nanospheres. As aforementioned, the miniemulsion tech-
nique allows the synthesis of well-defined nanoparticles with
controlled particle size and narrow size distribution by using
low amounts of surfactants, which are advantageous if the
particles are going to be used for coatings or biomedical ap-
plications. Recently the synthesis of surface phosphonate-
functionalized styrene and methyl methacrylate nanoparti-
cles was reported,[28] which were later used for cell uptake
and adhesive coatings for titanium implant materials.[5] To
this end, they used vinylphosphonic acid or vinylbenzylphos-
phonic acid as co-monomers in miniemulsion polymeri-
zations and additional surfactants were required to stabilize
the systems.
Similar surface phosphonate-functionalized nanoparticles
were applied as templates for the biomimetic mineralization
of hydroxyapatite (HAP).[18b] Once again, the desired func-
tionalities were incorporated by miniemulsion copolymeriza-
tion of phosphonic acid-containing co-monomers and addi-
tional surfactants. Phosphonate-functionalized particles are
very well-suited as templates for the formation of bone min-
eral. For example, organic bisphosphonates are already used
as physiological regulators for bone resorption in medicine
because they strongly bind to HAP crystals.[29]
The previous examples show that the exploitation of
phosphonate-containing surfmers would be highly beneficial
to functionalize the nanoparticles surface since it implies
working under surfactant-free conditions, causing less work
up, and also allowing for a better control in the particle and
distribution sizes.
A distinctive characteristic of miniemulsion polymeri-
zations is that, unlike emulsion polymerizations, the net dif-
fusion of compounds between individual droplets is sup-
pressed.[1,11] This difference between the two techniques also
implies that the requirements on the surfmer reactivity are
different. A surfmer that can be used in emulsion polymeri-
zation should have a low reactivity compared with the other
monomer(s), thus being incorporated at the final stages of
the polymerization.[17] This situation is important to avoid
the burial of the surfmer inside the particle while this one
grows during the polymerization due to the diffusion of
monomers, which may affect the stability of the final prod-
uct. However, a low reactivity of the surfmer would lead to
a low copolymerization with the other monomer. Converse-
ly, in miniemulsion processes the reactivity of surfmers
should be high (or comparable to the one of the other mon-
omers) to ensure their copolymerization on the surface of
the nanoparticle, because they are highly likely to be at and
react on the surface of the droplets, which do not change
sizes during the polymerization process. Thus, the duality of
acting simultaneously as surfactant and co-monomer is suc-
cessfully exploited in the miniemulsion polymerization to
generate surface-functionalized nanoparticles in a simple
polymerization reaction without the use of high amounts of
functionalized co-monomers, which would be distributed all
over the particles and not strictly on their surface;[18] even if
the co-monomer is too hydrophilic it could result in a homo-
polymerization forming a “hairy” layer around the parti-
cle,[19] affecting the particle size.
An additional benefit of the miniemulsion polymerization
is that the composition of the final colloids resembles the
composition of the dispersed monomer phase, thus defining
each droplet as a nanoreactor. Especially with regard to co-
polymerization of different co-monomers and crosslinkers
or embedding of dyes and drugs, this concept allows the
preparation of well-defined polymeric particles, meaning
that all incorporated functionalities are equally distributed
in each particle.[1,11] This represents an advantageous aspect
in our design because surfmers can be copolymerized by the
miniemulsion process to homogeneously functionalize the
nanoparticle surface.
In the cases in which surfmers contain at least one addi-
tional functional group, besides the polymerizable one, these
compounds are considered as functional surfmers. They are
of great interest since the introduction of tailored function-
alities onto the surface of the nanoparticles (NPs) without
any additional post-polymerization chemical modification is
easily achieved. Those functionalities may be exploited af-
terward for further chemical/physical functionalization of
the NPs toward specific applications. There are a few func-
tional surfmers reported in the literature possessing alco-
hol,[20] carboxylic,[21] active ester,[22] sulfate,[23] phospho-
Herein, we present the synthesis of novel phosphonate-
containing polymerizable surfactants bearing a methacryla-
mide group as polymerizable unit and a phosphonic acid as
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New Phosphonate Surfmers
FULL PAPER
polar head separated by a hydrophobic spacer, as shown in
Schemes 1 and 2. The exploitation of the synthesized com-
pounds in miniemulsion polymerizations of styrene and
methacrylates is also presented. Additionally, the first ever
fluorescent surfmer (Scheme 3) was synthesized and studied
as a model system to evaluate the copolymerization of
surfmers in miniemulsion polymerizations. Finally, applica-
tion studies of the phosphonate-functionalized nanoparticles
obtained using the previously synthesized surfmers were car-
ried out to evaluate the viability and applicability of these
systems. Specifically, on one hand, phosphonate-functional-
ized nanoparticles were successfully utilized to carry out cel-
lular-uptake experiments. And on the other hand, we effec-
tively performed surfmer-mediated biomimetic mineraliza-
tion of HAP by using the same surface-functionalized nano-
particles as templates.
Scheme 1. Synthesis route to C12-PET. a) phthalimide potassium salt,
DMF, 16 h at 808C; b) triethyl phosphite, 16 h reflux; c) hydrazine mono-
hydrate, EtOH, 30 min at 08C, 2.5 h reflux; d) methacryloyl chloride,
H2O, CH2Cl2, Na2CO3, 30 min at 08C, 3 h at RT; e) i) TMSBr, CH2Cl2,
3 h at RT; ii) MeOH, 3 h at RT.
Results and Discussion
Synthesis of the surfmers: Synthesis of C12-PET: To func-
tionalize a particle exclusively on the surface with phospho-
nate groups in a miniemulsion approach, a compound that
bears a polymerizable group and has a hydrophobic tail of
the appropriate length to balance the hydrophilicity of the
headgroup is needed. Taking the hydrophilic-lipophilic bal-
ance (HLB) theory into account a spacer with 12 -CH2-
units seemed to be the best choice to balance a phosphonate
headgroup. Also different authors have reported the high
surface activity of dodecylphosphonic acid and tetradecyl-
phosphonic acid.[30]
It has been reported that the position of the polymeriz-
able group in the molecule has a great influence on the co-
polymerization of the compound in an emulsion/miniemul-
sion polymerization process.[31] As a general rule, we consid-
er that it is more appropriate to locate the polymerizable
group at the very end of the hydrophobic segment because
it may be deeply immersed into the (hydrophobic) monomer
phase. We designed the incorporation of the polymerizable
moiety by an amide linkage, forming a methacrylamide. The
synthetic strategy was tailored in such a way that no addi-
tional steps for protection–deprotection of functional groups
were needed, and the sensitive polymerizable group could
be incorporated in one of the last steps, as shown in
Scheme 1.
12-Methacrylamidododecylphosphonic acid (C12-PET)
was synthesized starting from 1,12-dibromododecane, which
is an inexpensive compound and commercially accessible for
large scale synthesis.
To obtain compound 4 a combination of the Gabriel syn-
thesis with the Arbuzov reaction was designed. First, com-
pound 1 was asymmetrically monosubstituted using potassi-
um phthalimide by using the first step of the Gabriel synthe-
sis. The phthalimide group was used as a protecting group of
the already incorporated primary amine in compound 2,
which permitted us to perform the Arbuzov reaction to in-
corporate a phosphonic ester at the other end of the hydro-
carbonated chain. Triethylphosphite (PACTHNURTGNENG(U OEt)3) was used as
reagent and solvent, giving phosphonate 3 in excellent yield.
Among the different possibilities for the cleavage of the
phthalimide group, we considered that it is more convenient
to carry it out using hydrazine, what is also known as the
Ing–Manske variation of the Gabriel synthesis.[32] Thus, we
deprotected the amine group in compound 3 generating 4.
Amidation of compound 4 was performed with methacry-
loyl chloride to give compound 5 under Schotten–Baumann
conditions. Thus, the amidation was performed as a hetero-
phase reaction under basic conditions. The great benefit of
this heterophase reaction, consisting of dichloromethane
and water, is that an inorganic base could be used, which is
only soluble in water and therefore easy to remove once the
reaction is finished.
Deprotection of the phosphonic acid to finally generate
C12-PET was achieved by applying a modified method from
the one previously reported by McKenna and co-workers.[33]
In the first step a transesterification of the phosphonic acid
ester to the trimethylsilyl phosphonate is realized, by treat-
ing the ester with an excess of bromotrimethylsilane
(TMSBr). In contrast to the McKenna route, which consists
in using water for cleaving the freshly formed trimethylsilyl
phosphonate, methanolysis was our choice.
Synthesis of C11-PET: As predicted by the HLB-theory
C12-PET exhibited remarkably good properties in minie-
mulsions, as will be shown later in the miniemulsion poly-
merization section. However, the synthetic pathway only en-
abled very low yields of C12-PET to be obtained. Based on
these two facts, we designed a new synthetic approach, in
which the improvements were made on the first steps of the
synthesis. To synthesize C12-PET, the first step involved the
generation of an asymmetric compound, which led to a very
low yield of compound 2; this step is now replaced by the
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P. Froimowicz, K. Landfester et al.
ples, which were then measured by IR spectroscopy.[37] As
surfactants might be strongly adsorbed onto the particle sur-
face, which might lead to an incomplete replacement by the
aforementioned methods, we have chosen another approach
that gave us the opportunity to analyze and prove the cova-
lent attachment in a more reliable manner. We synthesized
a dye-containing surfmer, which could be straightforwardly
studied after polymerization by SEC overlapping both the
RI and UV chromatograms. The chemical structure is com-
parable to C12-PET and C11-PET. A fluorescent naphthali-
mide group was chosen as the label. The fluorogenic 4-
bromo-1,8-naphthalenedicarboxylic anhydride is commer-
cially available and could be chemically addressed straight-
forwardly at two different sites with high selectivity for its
functionalization.[38] The synthetic approach is presented in
Scheme 3.
Scheme 2. Synthesis of C11-PET. a) phthalimide potassium salt, DMF,
16 h at 808C; b) PBr3, toluene, 2.5 h at 1008C; c) triethyl phosphite, 16 h
reflux; d) hydrazine monohydrate, EtOH, 30 min at 08C, 2.5 h reflux;
e) methacryloyl chloride, H2O, CH2Cl2, Na2CO3, 30 min at 08C, 3 h at
RT; f) i) TMSBr, CH2Cl2, 3 h at RT; ii) MeOH, 3 h at RT.
two step synthesis of compound 8 (Scheme 2), which can be
considered as a synthetic equivalent of compound 2 because
the compounds only differ in one -CH2- unit. Even though
the new synthetic pathway included one more step, the pro-
cedures for synthesizing and purifying the compound were
much simpler and efficient, enabling a much higher overall
yield for compound 8 (61%) than for 2 (15%). The new
generated surfmer (C11-PET) would have a hydrophobic
spacer formed by a -(CH2)11- unit, which is expected not to
significantly change the properties exhibited by C12-PET
since the only difference between both compound is just
one -CH2- unit.
Specifically, substitution of the bromine in compound 6 by
phthalimide and subsequent bromination of the alcohol of
the freshly obtained compound 7 by using phosphorus tri-
bromide (PBr3) gave the designed product 8. Both products
7 and 8 were purified by recrystallization. From this point
on, all the following steps in the synthesis of surfmer C11-
PET were carried out as already described for C12-PET. As
a result of the new synthetic design the overall yield for
C12-PET (7%) was remarkably enhanced in the synthesis
of C11-PET (36%). Moreover, C11-PET was also subjected
to miniemulsion polymerizations showing no significant dif-
ferences with C12-PET, as will be shown and discussed in
the miniemulsion polymerization section.
Scheme 3. Synthetic strategy for Fluoro-PET: Synthesis of 11: a) triethyl
phosphite, 16 h reflux; b) hydrazine monohydrate, ethanol, 30 min at
08C, 2.5 h reflux. Synthesis of Fluoro-PET: c) compound 11, ethanol,
reflux, overnight; d) diaminodecane, dioxane, triethylamine, 60 h reflux;
e) methacryloyl chloride, water, CH2Cl2, methanol, Na2CO3, 30 min at
08C, 3 h at RT; f) i) TMSBr, DMF, overnight at RT; ii) MeOH, 3 h at RT.
Synthesis of Fluoro-PET: Surfmers are likely to be incorpo-
rated into the polymer backbone, however, a major chal-
lenge in this research field is how to demonstrate the cova-
lent attachment of the surfmer to the polymeric particles. A
great deal of work has been undertaken to try to evidence
the copolymerization in different studies. The authors moni-
tored the surface tension,[16,34] dialyzed the samples exten-
sively and measured the particle charge[35] or determined the
amount of surfactant in the supernatant after centrifugation
by using size-exclusion chromatography (SEC) and NMR
studies,[34a] or NMR spectroscopy on the latex itself by using
pulse sequences for water suppression.[13,31] Other proce-
dures included the serum replacement method and subse-
quent two-phase titration[36] or extensive dialysis of the sam-
The hydrophilic headgroup of the fluorescent surfmer
(phosphonic acid group) is linked to the fluorogenic system
by the formation of an imide bond and separated by
a -(CH2)3- spacer to form the imide 13. The anhydride group
in 12 was reacted with diethyl 3-aminopropylphosphonate
(11), which was beforehand synthesized in a two-step syn-
thesis from the commercially available compound 10, using
the Arbuzov reaction as well as the Ing–Manske variation of
the Gabriel synthesis.
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By means of a nucleophilic aromatic substitution, 1,10-di-
aminodecane was incorporated into the naphthalimide
moiety, replacing the bromine in position 4 to generate com-
pound 14. The polymerizability of the molecule was again
achieved by amidation of the free amine group in 14 with
methacryloyl chloride under Schotten–Baumann conditions,
obtaining 15 in a very good yield. In the final step, Fluoro-
PET was synthesized by cleaving the phosphonic esters in 15
using the same procedure as for C12-PET and C11-PET. All
increasing the ratio of C12-PET in the polymerizations pro-
duced diminution in the particle size of the synthesized
nanoparticles. Different monomers could be polymerized.
Both organic- and water-soluble initiators were demonstrat-
ed to initiate the free-radical polymerizations and the solid
content after polymerizations were always very close to the
theoretical values. All the results are presented in Table S1
in the Supporting Information. These results showed that
C12-PET behaved as a surfmer rather than as a conventional
co-monomer.
1
the synthesized products were analyzed by H, 13C, 31P NMR
spectroscopy and mass spectrometry. Moreover, the absorp-
tion and emission spectra were recorded for the fluorescent
compounds. Fluoro-PET was obtained as a yellow powder,
which is soluble in water under slightly basic conditions. The
absorption and emission spectra show only a slight overlap,
with an absorption maximum at 467 nm and emission maxi-
mum at 550 nm in water, with a Stokes shift of 83 nm. As
will be shown in the later sections, this compound shows the
desired characteristics, including absorbance, fluorescence,
polymerizability, and surface activity.
Figure 1 shows representative SEM images of the differ-
ent latexes synthesized using C12-PET as surfmer. It can be
seen from the pictures that all systems polymerized giving
narrow size distributions. Figure 1A and B show the change
As aforementioned, compound 12 can be selectively
modified at two different sites,[38] either by reacting the an-
hydride or substituting the bromine in position 4. The deci-
sion for the incorporation of the hydrophilic group in the
fluorophore by the formation of an imide was based mainly
on the final molecular geometry of the final Fluoro-PET,
since the naphthalimide group is highly conjugated and vol-
uminous compared with the other groups forming this
surfmer and also compared with the previously synthesized
C12-PET and C11-PET. Therefore, we considered it impor-
tant to orientate the more polar side of the chromophore
(the imide group) toward the same direction in which the
headgroup (phosphonic acid) was placed, thus leading to
a more realistic comparability between all the three surfm-
ers.
Miniemulsion polymerization: The synthesized compounds
were evaluated on their ability to stabilize miniemulsions.
Thus, different miniemulsion systems varying monomers,
ratio of surfmer and initiators were studied. The non-poly-
merizable surfactant sodium dodecylphosphonate (SDP)
was used as a reference in control experiments. If not other-
wise stated, in all cases the surfactant/surfmer solution used
to prepare the continuous phase was 0.01 mollꢀ1, for sim-
plicity reasons this concentration is referred to as the stan-
dard concentration. The number in front of the surfactant/
surfmer in the tables is a multiplication factor for the stan-
dard concentration, thus 2·C12-PET means that a solution
of 0.02 mollꢀ1 was used in that particular synthesis. All min-
iemulsions were filtered prior to analysis to eliminate any
interference from the very slight amount of coagulum pres-
ent in some samples.
Figure 1. SEM images of nanoparticles stabilized with C12-PET. A) poly-
styrene, initiator V59, 0.5 C12-PET; B) polystyrene, initiator V59,
1.0 C12-PET; C) polystyrene, initiator KPS, 1.0 C12-PET; D) polystyrene,
initiator KPS, 1.0 SDP; E) poly(n-butyl methacrylate), initiator KPS,
1.0 C12-PET. The number in front of the surfactant/surfmer is a multipli-
cation factor for the standard concentration (0.01 mollꢀ1).
in particle size varying the concentration of the surfmer
from 5 to 10 mmol. Lꢀ1, respectively, and the results were
confirmed by the dynamic light scattering (DLS) measure-
ments (see Table S1 in the Supporting Information). Both of
these systems were polymerized using V59 as a hydrophobic
initiator.
C12-PET as surfmer for miniemulsion polymerization: As
predicted by the HLB theory, C12-PET showed to be a very
effective surfmer since it was highly efficient stabilizing the
miniemulsion and resulting dispersions. Results showed that
Figure 1C shows a SEM image of a latex polymerized in
similar conditions as for the one shown in Figure 1B, but
when using the water-soluble KPS initiator we obtained no
significant characteristic differences between the systems
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P. Froimowicz, K. Landfester et al.
(see Table S1 in the Supporting Information). For compari-
son, nanoparticles prepared with the conventional surfactant
SDP and KPS are presented in Figure 1D. Figure 1E shows
particles of poly(n-butyl methacrylate) (PBMA). As PBMA
has a Tg slightly higher than room temperature, the particles
tend to melt under the electron beam and then they do not
look perfectly spherical.[39] However, it is still possible to
notice the nice narrow size distribution of the resulting
nanoparticles.
To study the covalently attachment of C12-PET to the
polymer backbone additional studies were carried out. We
designed a series of complementary NMR experiments to
study the presence or absence of the polymerizable metha-
crylamide belonging to C12-PET by studying the proton
spectra of the methacrylamide by 1H NMR spectroscopy
Supporting Information) was dialyzed, then 1 mL was
freeze-dried and the initial amount of C12-PET (as for PS-
2) was added, finally the mixture was re-suspended in 1 mL
[D6]DMSO (spectrum ii); 1 mL of a freshly prepared poly-
styrene dispersion polymerized using C12-PET (PS-2 in the
Supporting Information) was freeze-dried and re-suspended
in 1.0 mL [D6]DMSO (spectrum iii). Spectrum iii in Fig-
ure 1A shows that the signals from the methacrylamide pro-
tons belonging to the surfmer were not detectable for the
polymerized surfmer, whereas in the case of the mixture
(spectrum ii), used as reference, the signals of those protons
were clearly visible at d=5.6 and 5.3 ppm and are in good
accordance with the chemical shifts for the protons of the
surfmer, as evidenced by spectrum i, which corresponds to
pure C12-PET.
(H2C=C
G
A complementary control experiment to test the sensitivi-
ty of the previous NMR measurements was carried out by
performing 31P NMR analysis of the polymerized sample in
which C12-PET was used. To this end, we used the same
sample studied by 1H NMR spectroscopy and shown in
Figure 2 A, spectrum iii; the result is presented in Fig-
ure 2B. It can be seen that the signal at d=27.35 ppm be-
longing to the phosphonate groups is clearly observable.
The fact that this signal is broadened shows that the phos-
phonates have a limited mobility caused by their covalent
attachment to the particle surface. Thus, the complementary
To evaluate the polymerization of the surfmer three sam-
ples were prepared and studied by H NMR spectroscopy:
pure surfmer C12-PET (spectrum i); as a reference a poly-
styrene dispersion polymerized using SDP (see PS-1 in the
1
information collected upon H and 31P NMR measurements
shows that a) the surfmer can be easily detected and is
highly possible that is located on (or at least near) the sur-
face of the particle; and b) whereas the phosphonates indi-
cate their presence on the particle surface, the absence of
the proton signals belonging to the polymerizable moiety
strongly suggest that this has been copolymerized forming
part of the nanoparticle polymer backbone.
C11-PET and Fluoro-PET as surfmers for miniemulsion
polymerization: To study the influence in particle sizes and
to determine where the amount of surfactant is no longer
sufficient for the stabilization of the miniemulsions, a series
of miniemulsions were synthesized by using C11-PET and
Fluoro-PET with decreasing amounts of surfmer. As a mea-
sure for the stability and efficiency of the surfmer, the solid
content after filtration and a monomodal particle size distri-
bution were taken into account. The results are summarized
in Table 1 for C11-PET and Table 2 for Fluoro-PET.
As shown in the two tables, the Fluoro-PET miniemul-
sions seem to be more stable in this concentration range
since in the case of C11-PET the solid content is decreasing
when the surfmer amount is lowered under a certain level.
As can be seen from Table 1 and Table 2, the particle size
depends on the amount of surfmer used; the higher the
amount of surfmer, the smaller the particle sizes.
In addition, the hydrophilicity of the monomer seems to
affect the final particle size; thus, with the same amount of
surfmer, the particles are bigger for both C11-PET and
Fluoro-PET when using methyl methacrylate than for those
obtained using styrene.
Figure 2. A) 1H NMR spectra sections in [D6]DMSO: i) C12-PET, ii) dis-
persion stabilized with SDP and C12-PET added (PS-1), iii) dispersion
stabilized with C12-PET (PS-2). B) 31P NMR of PS-2, dialyzed, freeze-
dried and redispersed in [D6]DMSO.
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New Phosphonate Surfmers
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Table 1. Concentration series of C11-PET for stability test, for styrene
and methyl methacrylate polymerizations.
Samples[a]
Monomer Surf. ratio
DLS
Solid
content [%]
dh [nm]
s [nm]
PS-1s
PS-2s
PS-3s
PS-4s
PMMA-1s
PMMA-2s
PMMA-3s
PMMA-4s
St
St
St
St
MMA
MMA
MMA
MMA
2.0
1.0
0.5
0.25
2.0
1.0
0.5
0.25
140
204
280
315
201
336
384/553
378/520
19
23
30
37
52
36
32/55
34/48
21.5
19.7
14.7
12.2
20.1
20.4
18.5
15.3
Figure 3. SEM images of polystyrene nanoparticles polymerized with
C11-PET (A) and Fluoro-PET (B), corresponding to samples PS-2s and
PS-7s, respectively.
[a] In all samples the “s” refers to polymerization carried out in small
batches (see the Supporting Information for further details).
Table 3. SEC results for Fluoro-PET polymerizations of styrene and
methyl methacrylate.
Table 2. Concentration series of Fluoro-PET for stability test, for styrene
and methyl methacrylate polymerizations.
Sample
Mn
Mw
PDI
UV[a]
RI
UV[a]
RI
UV[a]
RI
Samples[a]
Monomer
Surf. ratio
DLS
Solid
PS-7s
PMMA-7s
Fluoro-PET
18ꢄ104
8.5ꢄ104
–
21ꢄ104
10ꢄ104
474
36ꢄ104
24ꢄ104
–
39ꢄ104
24ꢄ104
553
2.01
2.33
–
1.86
2.84
1.17
content [%]
dh [nm]
s [nm]
PS-6s
PS-7s
PS-8s
PS-9s
PMMA-6s
PMMA-7s
PMMA-8s
PMMA-9s
St
St
St
St
MMA
MMA
MMA
MMA
2.0
1.0
0.5
0.25
2.0
1.0
0.5
0.25
116
125
140
189
203
166
211
214
18
19
19
22
23
25
29
28
21.0
20.5
19.2
19.5
20.9
18.9
20.2
18.2
[a] In all samples the UV/Vis detector was always set at 440 nm, which
corresponds to the maximal absorption of Fluoro-PET.
Efficiency on the surface functionalization by using surfmers
versus monomers: The quantification of the phosphonate
groups on the particle surface was performed by means of
a polyelectrolyte titration with the oppositely charged poly-
electrolyte. A low molecular-weight polydiallyldimethylam-
monium chloride (polyDADMAC) was employed as poly-
electrolyte. The titration endpoint was determined as the
point of zero streaming potential, by using a particle charge
detector (PCD, Mꢃtek, Germany). The measurements were
conducted with diluted dispersions (0.1 wt%) at pH 10.8, to
ensure that all phosphonate groups are completely depro-
tonated in the aqueous solution (pK1 =2.6 and pK2 =7.3).
The amount of functional groups were calculated from the
consumed volume of polyelectrolyte, the solid content, the
particle size, and the polymer density according to a reported
methodology.[40] The results are summarized in Table 4 and
further results are provided in the supporting information in
Table S2 (see the Supporting Information).
[a] In all samples the “s” refers to polymerization carried out in small
batches (see the Supporting Information for further details).
The previous results show that C11-PET and Fluoro-PET
can also be viewed as effective surfmers since they were
highly efficient stabilizing the miniemulsion and the result-
ing dispersion. Figure 3 shows two representative images of
styrene nanoparticles obtained with C11-PET in Figure 3A
and Fluoro-PET in Figure 3B.
The covalent attachment of C11-PET to the polymer
backbone, however, was once again difficult to study, al-
though all the results led to the same conclusion as for C12-
PET, thus strongly suggesting its copolymerization. It is in
this context that Fluoro-PET was exploited to further study
the copolymerization of surfmers since, besides the NMR
experiments, it was also possible to perform SEC measure-
ments by using both an RI and UV/Vis detector;
the latter was set up at 440 nm corresponding to the
Table 4. PCD results for the miniemulsion polymerizations carried out using the dif-
ferent surfmers and SDP.
maximal absorption of Fluoro-PET. The results for
a PS and a PMMA system are shown in Table 3,
which also shows the SEC result for pure Fluoro-
PET.
Samples Surf.
type
dh
-PO32ꢀ groups
[nm][a]
Per g of Per g of Per g of Max. Theo.
Incorp.
polymer polymer polymer per g of poly- efficiency
An additional feature of the latexes obtained by
copolymerization of Fluoro-PET is that they were
fluorescent in all the cases. Although no quantifica-
tion was carried out, this would strongly indicate
that no complete self-quenching happened between
the Fluoro-PET molecules, or at least it was not
a dominant effect. Thus, a homogeneous distribu-
tion of the surfmer on the particle surface is as-
sumed.
ꢄ1018
ꢄ1018
ꢄ1018
mer ꢄ1018
PS-4
PS-5
SDP
C12-
PET
C11-
PET
98
107
11.0
27.3
0.57
1.83
0.19
0.51
–
50.0
–
54
PS-6
118
26.0
47.5
2.00
4.96
0.51
1.03
50.0
50.0
52
95
PS-6s
Fluoro- 124
PET
[a] These values correspond to dialyzed samples.
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P. Froimowicz, K. Landfester et al.
Table 4 shows that the use of surfmers leads to a much
higher number of charges on the particle surface. Even
though the samples with surfmer were much longer dialyzed
than the SDP samples, there was no lack of dispersion sta-
bility. Moreover, C11-PET and C12-PET behaved very simi-
larly; not only producing particles in the same size range
but also introduced approximately the same amount of
-PO32ꢀ groups on the surface of the particles.
We have recently reported the functionalization of nano-
particles with phosphonate groups through copolymerization
of conventional monomers with either vinylphosphonic acid
(VPA) or vinylbenzylphosphonic acid (VBPA).[28a] The
amount of functional groups was extremely dependent on
the hydrophilicity of the monomer. Whereas the functionali-
zation density with VPA (as the more hydrophilic mono-
mer) is rather poor, VBPA led to much higher functionaliza-
tion densities. If the amount of phosphonate groups is calcu-
lated on the mass of polymer, 1% of VBPA led to almost
the same functionalization density as when the same molar
amount of C12-PET is used. In the latter case, however,
much smaller particles are obtained, which may be benefi-
cial in the suggested application for the coating of titania
implant materials since the packing on surfaces would be
more dense with smaller particles.
tained synthesized latexes were investigated. Since the nano-
particles were dye-labeled, confocal laser scanning microsco-
py (CLSM) was also applied to gain better insights into the
uptake. To this end, the cells were treated with 75 mgmLꢀ1
of fluorescent-labeled nanoparticles for 20 h and both the
particle uptake and the cytotoxicity were quantified by flow
cytometry. The number of dead cells (cytotoxicity) was
quantified with the fluorescent DNA-intercalating dye 7-
AAD, which can enter only into dead cells with disintegrat-
ed membranes.[43]
Figure 4A shows the HeLa cells uptake results obtained
for phosphonate-functionalized polystyrene particles poly-
merized using C12-PET and SDP. The experiments were
carried out with dialyzed and non-dialyzed samples of each
latex. Thus, PS-4_d and PS-4_nd correspond to dialyzed and
non-dialyzed nanoparticles polymerized using the conven-
tional SDP, whereas PS-5_d and PS-5_nd correspond to
surfmer-polymerized nanoparticle dialyzed and non-dia-
lyzed, respectively, (for a detailed particle description see
Table S1 in the Supporting Information).
Application studies of the phosphonate-functionalized nano-
particles: As already mentioned in the introduction, phos-
phonic acids have a variety of applications, but only little is
known about nanoparticles that are surface-functionalized
with phosphonic acid groups. As the miniemulsion process
would also allow the embedding of drugs and growth factors
into nanoparticles, we investigated the viability and applica-
bility of the functionalized nanoparticles as templates for
the biomimetic mineralization and also performed cellular-
uptake experiments to evaluate their use in biological sys-
tems.
Cellular uptake and cytotoxicity of phosphonate-functional-
ized nanoparticles: Several studies on the interactions be-
tween different cell types and nanoparticles presenting dif-
ferent sizes, shapes, and surface functionalization have been
reported.[19a,41] For instance, we have highlighted how benefi-
cial the exploitation of phosphonic acids on the particle sur-
face is toward biological application, such as cellular uptake
experiments[42] and coatings for implant materials.[18a] In
those studies, the particles were obtained by the copolymeri-
zation of synthetic co-monomers using surfactants. Herein,
we present preliminary results showing the successful exploi-
tation of phosphonate-functionalized nanoparticles generat-
ed by miniemulsion polymerization under surfactant-free
conditions by using the previously synthesized surfmers in
cellular-uptake experiments.
Figure 4. Uptake and viability of phosphonate-functionalized nanoparti-
cles: PS-4_d and PS-4_nd corresponding to dialyzed and non-dialyzed
nanoparticles polymerized using the conventional SDP, and PS-5_d and
PS-5_nd corresponding to surfmer-polymerized nanoparticle dialyzed
and non-dialyzed. HeLa cells were incubated with 75 mgmLꢀ1 fluores-
cently (PMI) labeled nanoparticles for 20 h and both particle uptake (A)
and cytotoxicity (B) were analyzed by flow cytometry. Particle cell
uptake was normalized to the fluorescence value of a standard polystyr-
ene nanoparticle and expressed as normalized median fluorescence inten-
sity (rfu; relative fluorescence units). Mean values and standard devia-
tions were calculated from two independent experiments which were run
in triplicate.
Specifically, two types of human cells, a cervix cancer cell
line (HeLa) and human mesenchymal stem cells (MSCs)
were studied. Using both surfmers (C12-PET and C11-PET)
and the conventional SDP surfactant (as a reference), not
only the cellular uptake but also the cytotoxicity of the ob-
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New Phosphonate Surfmers
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Phosphonate-functionalized nanoparticles showed similar
uptake rates in HeLa cells for the analyzed samples with ex-
emption of the non-dialyzed SDP stabilized particles PS-
4_nd, which experienced about 30% higher uptake ratio
than its corresponding dialyzed PS-4_d. It is worth noting
that although a higher surfactant (SDP) concentration
seems to favor the cellular uptake, the result clearly eviden-
ces a strong dependence on the purification level (see the
Experimental Section). This inconvenience is not observed
in the surfmer-polymerized samples, in which the cell
uptake ratios were similar regardless of the dialysis of the
samples.
Figure 4B shows the results for the viability analysis mea-
sured by flow cytometry for the aforementioned samples,
namely PS-4_d, PS-4_nd, PS-5_d, and PS-5_nd. It can be
seen from this Figure that whereas the dialyzed nanoparti-
cles presented a negligible cytotoxicity (ꢁ5% apoptotic
cells), the non-dialyzed samples exhibited a very small toxic
effect (ꢁ10% apoptotic cells). These results were independ-
ent on the use of C12-PET or SDP.
These results are in accordance with other studies, in
which phosphonate-functionalized particles showed a good
uptake in dendritic[44] and mesenchymal stem cells[5] without
a significant damage on the viability. Phosphonate-function-
alized particles also do not impair cell proliferation and ac-
tivity.[5,45]
Apatite growth on the particle surface of phosphonate-func-
tionalized nanoparticles: We have previously reported that
phosphonate-functionalized particles of about 200 nm were
successfully mineralized in dispersion or adhered to a titani-
um substrate[18] It was found that it was critical to synthesize
smaller surface-functionalized nanoparticles without using
large amounts of non-ionic surfactant nor SDS as surfactant,
which showed to counteract an effective loading with miner-
als,[2] resulting in only very small amounts of hydroxyapatite
(HAP) crystals on the particle surface. The low amount of
crystals found on the particle surface was explained by a pos-
sible shielding effect of the functional groups by excess of
non-ionic surfactant or small residues of SDS that could not
be removed from the particle surface in the washing step.[2]
Herein, we have demonstrated that by using a surfmer for
the synthesis of small functionalized particles these prob-
lems were circumvented. Phosphonate-functionalized parti-
cles of around 100 nm size were synthesized through a surfm-
er-approach and applied to grow HAP on the particle sur-
face. The morphology of the untreated particles were al-
ready shown in Figure 3A.
The human MSCs showed uptake rates of approximately
one order of magnitude lower than HeLa cells for the same
set of studied samples (see the Supporting Information, Fig-
ure S1).
The exploitation of C11-PET as surfmer was also investi-
gated for these cell-uptake experiments showing no differen-
ces when compared to the results obtained for C12-PET
(see the Supporting Information).
Finally, Figure 5 shows confocal live-cell images of HeLa
cells after the uptake of different nanoparticles (which
appear as green in the images due to dye used to label
them). Figure 5A shows the uptake results using PS-5_d,
whereas Figure 5B shows the results obtained for PS-6_d
(which was synthesized using C11-PET, see Table S1 in the
Supporting Information). Additional and comprehensive
CLSM images of the uptake experiments with different
surfmer-functionalized and SDP-stabilized particles in HeLa
and human MSCs are shown in Figure S2 in the Supporting
Information.
Mineralization onto the surface of the functionalized par-
ticles was carried out following an already reported method-
ology,[18b] basically consisting on the sequential loading of
the surface with the respective ions. Once the process was
finished, the particles were homogeneously covered with
crystals as it can be seen in the SEM overview image (Fig-
ure 6A). In the enlarged TEM image (Figure 6B) numerous
Figure 6. SEM (A) and TEM (B) images showing the formation of HAP
crystals on the surface of phosphonate-functionalized nanoparticles pre-
pared with C11-PET.
crystals attached to the particles are clearly visible. To main-
tain the colloidal stability of the dispersion during the crys-
tallization process, very small amounts of a nonionic surfac-
tant (Lutensol AT50) were added before the loading. XRD
revealed that the crystals formed on the surface of the parti-
cles are indeed hydroxyapatite (see the Supporting Informa-
tion, Figure S3). The crystalline nature of the HPA was addi-
tionally confirmed by using dark field TEM images (shown
Figure 5. Confocal live cell images of HeLa cells incubated with
75 mgmLꢀ1 of latexes PS-5_d (A) and PS-6_d (B) for 20 h. The cell mem-
brane was stained with CellMask Orange (appears red), the nucleus was
stained with DraQ5 (appears blue), and the particles were labeled with
PMI (appears green).
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P. Froimowicz, K. Landfester et al.
in the Supporting Information, Figure S4). The thermogravi-
metric analysis of the sample showed that the percentage of
inorganic material in the sample is 32.9%, which is in
accord with the theoretical value of 33.3% (see the Support-
ing Information, Figure S5).
particles, from their design, synthesis and functionalization,
to their final applications.
Experimental Section
Synthesis of the surfmers and miniemulsion polymerizations: The entire
synthesis and characterization of each surfmer, as well as all miniemul-
sion polymerizations are fully described in the Supporting Information.
Conclusion
Methods: H, 13C, and 31P spectra were recorded on Bruker Avance spec-
In this paper we described the synthesis of three new surfm-
ers for miniemulsion polymerizations and employed them in
the polymerization of different monomers. All surfmers
were successfully copolymerized, without using any addi-
tional surfactants and generating very stable dispersions
(even after several months of storage), with a narrow parti-
cle size distribution. One of the synthesized surfmers
(Fluoro-PET) bears a fluorescent label and is the first dye-
labeled surfmer. The use of this Fluoro-PET permitted us to
study and evaluate the copolymerization between the surfm-
er and the other monomers by studying the resulting poly-
mers by using SEC with both the RI and the UV-detector
(set up at 440 nm corresponding to the maximal absorption
of Fluoro-PET) simultaneously. The excellent agreement
between the UV and RI chromatograms of the polymer-
forming particle confirmed the successful copolymerization.
This is a new approach that contributes to the long discussed
field of copolymerization of surfmers.
Regarding the application studies of these phosphonate-
functionalized nanoparticles, two preliminaries studies were
carried out: cell uptake of the functionalized nanoparticles
and biomimetic mineralization of hydroxyapatite on their
surface. On one hand, the favorable affinity of phosphonate
groups on the surface of the nanoparticles toward two differ-
ent cell types, a cervix cancer line (HeLa) and a human mes-
enchymal stem cell (MSC), was exploited to study their
uptake without significant cytotoxicity. Thus, we demonstrat-
ed here that surfmers provide a new area for the develop-
ment of new functionalized nanoparticles, which can be ob-
tained by miniemulsion polymerization with good intracellu-
lar uptakes rates for drug release in target cells, or for devel-
oping tailored surfmers toward metal prostheses with good
biocompatibility. On the other hand, the same phosphonate-
functionalized nanoparticles were used as functional scaf-
folds allowing the binding of calcium ions to subsequently
mineralize hydroxyapatite on the surface of the particles.
The results showed a high density of crystal grown on the
particle surface due to the high and uniform decoration of
the surface with phosphonic acid groups, which was provid-
ed by the copolymerization of the surfmers.
1
trometers with 250, 300, or 500 MHz. For 1H- and 13C NMR spectra tetra-
methylsilane was used as an external standard. In the case of 31P NMR
spectra, triphenylphosphine was employed as an external reference; the
signal of triphenylphosphine was set at d=ꢀ6.0 ppm.
Purification and characterization of the functionalized polymer nanopar-
ticles: All latexes were purified by dialysis. To evaluate the efficiency of
the purification, samples were measured at different dialysis time. As
a result, latexes were dialyzed for at least six days, with a daily water
change.
Surface tension was determined by the DuNoꢃy ring method at 208C
with a DCAT 21 device (Dataphysics, Filderstadt, Germany). All values
presented were averaged over ten repetitions of push–pull cycles. The
average hydrodynamic diameter DH of the particles and the particle size
distribution were measured by dynamic light scattering (DLS) (photon
cross-correlation spectroscopy PCCS) using a Nanophox PCCS (Sympa-
tec GmbH, Clausthal-Zellerfeld, Germany) at an scattering angle of 908
and a temperature of 258C. Dispersions were diluted to approximately
0.1 wt% with distilled water. The measurement parameters were set to
a count rate of 200 kcps with a measuring time of 100 s for each run and
three repetitions were conducted. The raw data was plotted in origin and
a gauss fit was done. Scanning electron microscopy (SEM) images were
taken with a Gemini 1530 (Carl Zeiss AG, Oberkochen, Germany). The
samples were prepared by drop casting of 0.01 wt.% dispersions on sili-
con wafers. The density of the functional groups on the particle surface
was determined by titration against the oppositely charged polyelectro-
lyte poly(diallyldimethyl ammonium chloride) (PDADMAC) using a par-
ticle charge detector (Mꢃtek GmbH, Germany) in combination with a Ti-
trino Automatic Titrator (Metrohm AG, Switzerland). The measurements
were conducted on 10 mL of the latex sample with a solid content of
1 mgmLꢀ1. The number of groups was calculated from the polyelectrolyte
consumption according to a methodology already reported.[40]
Mineralization of phosphonate-functionalized particles: The loading of
the particles was performed at 378C under stirring. The stability of the
particle dispersion was provided by adding 0.8 wt% nonionic surfactant
Lutensol AT50-containing aqueous solution by multiple centrifugation
and redispersion. First, the pH of all solutions was adjusted with a 28%
ammonia solution. Then, CaACHTUNRGTNEGNU(NO3)2 (0.5 mmol) was added to polymer
particles (ꢁ0.1 g) and stirred for 2 h to allow the binding of calcium ions
to the particle surface. Afterwards an aqueous solution of (NH4)2HPO4
(0.3 mmol) was added drop-wise during 1 h. After the loading process,
the samples were stirred for about 24 h. All the loadings were performed
at a constant molar ratio of calcium to phosphate ions of 5:3. The pH of
the particle dispersion was kept constant at pH 10 during the whole load-
ing experiment. The loaded samples were washed and freeze-dried
before XRD and TGA measurements. X-ray diffraction (XRD) was car-
ried out with a Phillips Typ PW diffractometer with Cu-Ka radiation (l=
1.54 ꢅ), 40 kV voltage, and 30 mA current.
It is worth mentioning that the herein-described surfmer
approach can effectively and controllably produce smaller
particles than the conventional approach using co-mono-
mers, and has no need for additional surfactants that might
cause difficulties in further envisaged applications.
Finally, we expect this functional surfmer approach, in-
cluding their potential applicability in the different research
areas, to greatly contribute to the field of functional nano-
Cellular uptake and cytotoxicity: Cell culture: Human cervix carcinoma
cells (HeLa) were kept in Dulbeccoꢆs modified eagle medium (DMEM),
supplemented with FCS (10%), penicillin (100 units) and streptomycin
(100 mgmLꢀ1), and l-glutamine (2ꢄ10ꢀ3 m) (all from Invitrogen, Germa-
ny). Cells were grown in a humidified incubator at 378C and 5% CO2.
For the nanoparticle uptake and the cytotoxicity measurements, adherent
HeLa cells were seeded at a density of 15,000 cells cmꢀ2 in 6-well-plates
(Greiner, Germany). On the following day, fluorescent particles (labeled
with PMI) were added at a concentration of 75 mgmLꢀ1 to the media in
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New Phosphonate Surfmers
FULL PAPER
the 6-well-plates without using a transfection agent. On the third day, ad-
herent cells in the 6-well-plates were detached in 2.5% trypsin (Gibco,
Germany), washed with magnesium- and calcium-free phosphate buf-
fered saline (PBS, Gibco, Germany), incubated with 7-aminoactinomycin
(7-AAD, 28.6 mgmLꢀ1) for 15 min at room temperature for the analysis
of cell viability and centrifuged. The pellet was resuspended in PBS for
analysis by flow cytometry.
Leica Laser Scanning Confocal Microscope, Leica, Germany), consisting
of inverse fluorescence microscope DMI 6000 CS equipped with a multi-
laser combination and with five detectors operating in the range of 400–
800 nm. A HCX PL APO CS 63ꢄ1.4 oil objective was used for these
studies. The fluorescent particles were excited with the argon laser (
ꢁ5 mW, l=488 nm), detected at 510–550 nm and are pseudo-colored in
green. Cell membrane was stained with CellMask Orange (2.5 mgmLꢀ1
,
For the dilution series of the surfactants SDS and SDP, 30,000 cells cmꢀ2
HeLa cells were seeded in 96-well-plates (Corning, USA). The surfac-
tants were solved in H2O and serial dilutions in DMEM with FCS (10%)
were performed. After 24 h, the cells were treated with the indicated sur-
factant concentrations and incubated for 20 h in a humidified incubator
(378C, 5% CO2). The live/dead staining were directly performed in the
96-well-plates with a final concentration of 1 mm calcein-AM (Invitrogen)
and propidium iodide (40 mgmLꢀ1; Fluka, Germany) for 30 min at room
temperature in the dark. Subsequently, the fluorescently stained cells
were measured with a fluorescence plate reader (Infinite M1000, Tecan,
Germany), excited with 490 nm, and detected at 530 nm for calcein, and
excited with 530 nm and detected at 645 nm for propidium iodide, using
i-control software (Tecan, Germany).
Invitrogen, Germany), excited with DPSS 561 nm (ꢁ20 mW), detected
at 580–620 nm. This signal from the cell membrane was pseudo-colored
in red surrounding the cytoplasm. The cell nucleus was stained with
DraQ5 (2.5ꢄ10ꢀ6 m, Biostatus, UK), excited with HeNe-laser (633 nm,
ꢁ10 mW), detected at 680–750 nm and appears in blue.
Acknowledgements
We thank Gunnar Glaser for support with the electron microscopy, and
Hansjçrg Menges for fluorescence measurements and studies.
Flow cytometry analysis: For the analysis of cell viability and the quan-
tification of cellular uptake of the nanoparticles, flow cytometry was
used. Flow cytometry measurements were performed with a CyFlow ML
using FlowMax 2.57 software (Partec, Germany). The FL1 channel
(527 nm) was used to analyze the uptake of nanoparticles, which were ex-
cited with 488 nm, and FL6 (675 nm) was used for 7-AAD measure-
ments, which were excited with 561 nm. For analysis, cells were selected
on a forward scatter/sideward scatter plot, thereby excluding cell debris.
These gated events were further analyzed for the FL1 and FL6 channels.
The median in FL1 was determined by analysis of 1D histograms. This
demonstrates the amount of nanoparticles taken up or associated with in-
dividual cells. For 7-AAD, the events in the cell gate were analyzed on
a FL1/FL6 dot-plot and three different populations (viable, apoptotic, or
dead) were determined by using negative controls and the apoptotic and
dead cells present in cell cultures. All values are triplicates of two inde-
pendent experiments with standard deviation. To normalize the median
fluorescence values from the flow cytometry measurement and to com-
pare the values of all particles it is necessary to introduce the factor of
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nFLPR ¼ FLIPR Px=FLIPR
ð1Þ
ð2Þ
PS
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Received: October 15, 2011
Published online: March 29, 2012
5212
www.chemeurj.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 5201 – 5212