Functionalised, photostable, fluorescent polystyrene nanoparticles of narrow size-distribution

Article abstract of DOI:10.1016/j.jphotochem.2011.11.012

Fluorescent nanoparticles continue to be of wide interest, as they have many advantages over single fluorescent molecules for biological imaging and sensing applications, such as increased fluorescence intensity and reduced photobleaching. In the followin

Full text of DOI:10.1016/j.jphotochem.2011.11.012

Journal of Photochemistry and Photobiology A: Chemistry 228 (2012) 60–67
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Functionalised, photostable, fluorescent polystyrene nanoparticles of narrow
size-distribution
Michal Pellach, Jenny Goldshtein, Ofra Ziv-Polat, Shlomo Margel∗
Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescent nanoparticles continue to be of wide interest, as they have many advantages over single
fluorescent molecules for biological imaging and sensing applications, such as increased fluorescence
intensity and reduced photobleaching. In the following work, styrene was copolymerised with a newly
synthesised, fluorescein-based, vinylic crosslinking monomer, by emulsion polymerisation, to create a
series of different sized fluorescent nanoparticles (35–100 nm), each of narrow size-distribution. The
particles were found to be highly fluorescent and with lower photobleaching compared to fluores-
cein isothiocyanate (FITC), offering an attractive alternative. The fluorescence excitation and emission
spectra were recorded, being similar to fluorescein, but with interesting variation in the excitation spec-
tra. The particles also have a wide range of potential uses, such as examining particle uptake activity
of a macrophage cell line, also demonstrated. The nanoparticles were coated with albumin to provide
functionality for potential conjugation to biological targeting agents.
Received 12 May 2011
Received in revised form
20 November 2011
Accepted 24 November 2011
Available online 13 December 2011
Keywords:
Fluorescent nanoparticles
Polystyrene nanoparticles
Fluorescein
Emulsion polymerisation
© 2011 Published by Elsevier B.V.
1. Introduction
into fibroblast cells [7]; fluorescent nanoparticles have been syn-
thesised using a perylene-based fluorescent monomer [1] as well
Fluorescent nanoparticles have attracted much interest in
biological imaging, biosensing [1], fluorescent cell sorting and
bioanalysis [2] due to their various advantages over single dye
molecules, such as increased fluorescence and reduced photo-
bleaching [3,4]. Besides the simple additive effect achieved by
confining a large number of fluorescent molecules into a small
volume, increasing the fluorescence per particle, dye molecules
entrapped within matrix particles have a matrix shielding effect
[5] from molecular oxygen, which reduces formation of reactive
oxygen species (ROS) hence reducing photobleaching. The matrix
also reduces mobility of the dye molecules reducing the chance of
collision, thus reducing fluorescence quenching [4], giving overall
increased photostability.
Covalently incorporating dye into particles rather than dye-
doping, greatly reduces potential problems such as dye aggregation
as well as leakage of dye from within the particles [6], and several
research groups have applied this technique. Brambilla et al. syn-
thesised rhodamine B cyanoacetate and covalently incorporated
the dye within poly(alkyl cyanoacrylate) nanoparticles, shown to
be appropriate for in vitro imaging of human brain endothelial cells
[6]; mesoporous silica nanoparticles with covalently attached FITC
have been prepared, and the particles were successfully uptaken
as bifunctional crosslinker [8]; nanoparticles as potential optical
imaging contrast agents have been synthesised, and polystyrene
microspheres have also been created, using fluorescent monomers
such as fluorescein-based or carbocyanine-based monomers
or 4-ethoxyl-N-allyl-1,8-naphthalimide, covalently incorporated
within the polymeric chains [9,10]. If fluorophores are located
within energy-transfer distance to each other, this may lead to
quenching [9], however, covalently attaching the dyes to other
monomers gives more control over distances between fluorescent
moieties.
Fluorescein, and fluorescein isothiocyanate (FITC) are widely
used for fluorescence, including biological targeting and imag-
ing, due to their wide accessibility, high quantum yield and FDA
approval for in vivo use of fluorescein [11–13]. However, its use
may be limited due to photobleaching [14,15], the kinetics of which
has been studied by Song et al. [16], as well as the limited flu-
orescent signals obtained from single fluorescent molecules in
biological labelling. By incorporating fluorescein into nanoparti-
cles, its advantages can be maintained while minimising effects of
bleaching, by slightly modifying the fluorescein molecule. One of
the reasons for fluorescein being so prone to photobleaching is the
vulnerability of the two phenol groups, which are highly suscepti-
ble to attack by radical ROS to form phenoxy radicals [17,18], which
can lead to destruction of aromaticity. Therefore, by exchanging
the hydroxyl groups with an alternative group photobleaching is
expected to be reduced.
∗
Corresponding author. Tel.: +972 3 5318861; fax: +972 3 5351250.
E-mail address: shlomo.margel@mail.biu.ac.il (S. Margel).
1010-6030/$ – see front matter © 2011 Published by Elsevier B.V.
doi:10.1016/j.jphotochem.2011.11.012

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61
A common method for synthesis of polymeric nanoparticles
with narrow size-distribution is by radical emulsion polymerisa-
tion. The emulsion polymerisation process begins with the minute
amount of insoluble monomer found in the continuous aqueous
phase, which, as the oligomeric chain grows, becomes even less
soluble and migrates into the surfactant micelles where polymeri-
sation continues. Further oligomeric chains enter between the
existing polymeric chains, and again, their polymerisation con-
tinues until termination [19]. Controlling factors such as total
monomer concentrations, crosslinker concentrations and surfac-
tant concentrations result in variation of the average nanoparticle
size [20–23].
The nanoparticles in the following work were prepared by
copolymerising a new fluorescent vinylic crosslinking monomer,
fluorescein-O,O-bis-methylstyrene (FBMS), with styrene by emul-
sion polymerisation. The resulting polystyrene-co-fluorescein-
O,O-bis-methylstyrene (PS-FBMS) nanoparticles synthesised are
intensely fluorescent, photostable, and have potential to be
exploited in many applications. Here we demonstrated their use
in examining particle uptake by a macrophage cell line. We also
coated the PS-FBMS particles with albumin, which provided amine
and carboxylate functional groups suitable for conjugation of a
bioactive agent.
III Tof/Tof Bruker, Germany) and elemental analysis (EA1110, CE
Instruments).
1H NMR (700 MHz, CD3OD): ıH 8.30 (1H, 19), 7.81 (1H, 17), 7.77
(1H, 18), 7.50 (2H, 34a,b), 7.46 (2H, 33a,b), 7.34 (1H, 16), 7.13 (2H,
26a,b), 7.00 (1H, 8), 6.97 (1H, 11), 6.96 (1H, 1), 6.93 (1H, 10), 6.75
(1H, 28), 6.71 (2H, 25a,b), 6.70 (1H, 36), 6.53 (1H, 2), 6.28 (1H, 4),
5.81 (1H, 29Z), 5.78 (1H, 37Z), 5.28 (1H, 37E), 5.25 (1H, 29E), 5.25
(2H, 31), 4.82 and 4.87 (2H, 23).
13C NMR (175 MHz, CD3OD): ıC 187.20 (3), 166.72 (21), 165.72
(9), 161.17 (5), 155.99 (7), 155.51 (13), 139.30 and 139.28 (27 and
35), 137.75(36), 137.69(28), 136.96(32), 135.06and134.92(15and
24), 134.08 (17), 132.51 (11), 132.41 (19), 131.71 (20), 131.69 (16),
131.31 (18), 130.75 (1), 129.71 (25a,b), 129.41 (2), 129.02 (33a,b),
127.61 (34a,b), 127.47 (26a,b), 118.26 (14), 116.31 (12), 116.25 (10),
115.04 (37), 114.71 (29), 105.57 (4), 102.59 (8), 71.73 (31), 68.41
(23).
HRMS (M+, C38H28O5) calculated: 564.63; found: 565.2010. Ele-
mental analysis: calculated C 80.83; H 5.00; O 14.17%; found C
80.86; H 5.01; O 14.11%.
2.3. Synthesis of fluorescent nanoparticles
The fluorescent nanoparticles were synthesised by emulsion
polymerisation, with varying ratios of FBMS to styrene, initiator
to monomer ratios and total monomer to continuous phase ratios.
In a typical experiment, FBMS (10 mg) was dissolved in styrene
(1.1 mL) and added to a deaerated solution of potassium persul-
phate (10 mg) and SDS (50 mg) in water (10 mL). The mixture was
polymerised overnight at 73◦, to yield nanoparticles of 60 10 nm
in diameter. While the mixture appeared uniform, any unpoly-
merised styrene was removed by disposal of the upper part of
the mixture, using a separating funnel. The particles were then
passed through a 0.45 ␮m filter, to remove unpolymerised FBMS,
and excess SDS as well as any small amount of monomers remain-
ing in solution were then removed by dialysis. Particle size was
varied by manipulation and control of concentrations of both SDS
and FBMS.
2. Materials and methods
2.1. Materials
The following analytical-grade chemicals were purchased from
Sigma–Aldrich and used without further purification: fluores-
cein (95%), p-chloromethyl styrene (97%), tetrahydrofuran (THF,
99.9%), (Bu)4NOH (40% in H2O), chloroform (CHCl3 99%), potas-
sium persulphate (99%), sodium dodecyl sulphate (SDS), styrene
(99%), FITC, and BSA. Water was purified by passing deionised
water through an Elgastat Spectrum reverse osmosis system (Elga,
High Wycombe, UK). Fluorescence intensity measurements and
absorbance measurements were performed using a microplate
reader with excitation wavelength 485 nm and emission wave-
length 535 nm (Spectrafluor Plus, Tecan, Männedorf, Germany).
An immunoperoxidase assay kit for determination of albumin in
human sera (HSA) (Immunology Consultants Laboratory, Inc.) was
used for qualitative determination of BSA. The cell line RAW264
was obtained from ATCC (VA, USA). DMEM, FCS, l-glutamine, peni-
cillin and streptomycin were purchased from Biological Industries,
Beit Haemek, Israel.
2.4. Particle characterisation
The hydrodynamic diameter and size distribution of the
nanoparticles dispersed in water were determined using a sub-
micron particle analyzer, model N4 Plus, Coulter Electronics Ltd.,
England. Excitation and emission spectra were measured by a spec-
trofluorometer (Cary Eclipse).
Thermogravimetric analysis (TGA) and differential scanning
calorimetery (DSC) were performed under nitrogen gas, by
TGA/DSC STARe System (Mettler Toledo, Switzerland).
2.2. Synthesis of FBMS
Synthesis was carried out according to a similar procedure
described in the literature [24]. Briefly, fluorescein (0.33 g, 1 mmol)
was dissolved in THF (7 mL). A 40% aqueous solution of (Bu)4NOH
was added to the solution and the reaction mixture was stirred
at room temperature for 2 h, after which the reaction solvent
was evaporated under vacuum. The dry product was dissolved
in CHCl3 (5 mL) and the reaction mixture was cooled to 5 ◦C. p-
Chloromethylstyrene (0.25 mL, 2 mmol) was added dropwise at
temperatures below 5 ◦C, and the mixture was maintained below
5 ◦C for 1 h, and then at room temperature for 18 h. Distilled water
(10 mL) was added and the product solution was extracted. The
product was precipitated from the CHCl3 solution then purified by
silica gel chromatography, and its structure was confirmed by NMR
(1H, 13C and 2D, Bruker Avance III–700), low resolution ESI pos-
itive mass spectroscopy (QTOF micro, “waters”, UK), MALDI High
Resolution Mass spectroscopy (using DHB matrix) with external
standard of (PEG/OMe)750, Reflection mode, YAG Laser (AutoFlex
2.5. Photobleaching experiment
An aqueous solution of FITC (0.01 ␮M, 100 ␮L) was prepared,
as was a dilute aqueous suspension of PS-FBMS nanoparticles, to
give similar fluorescence intensity (FI), at excitation wavelength
460 nm, and emission wavelength of 523 nm (0.02 0.005 mg/mL,
100 ␮L). The excitation slit was open to 20 nm and the emission
slit was open to 5 nm. Each of the samples was illuminated contin-
uously, and FI was measured over a period of twenty minutes by
a Cary Eclipse fluorescence spectrophotometer (Agilent Technolo-
gies, Inc.).
2.6. Cytotoxicity assay
96-Well plates were seeded with 2 × 105 cells per well of the
phagocyte cell line RAW264 in 100 ␮L DMEM supplemented with

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10% foetal calf serum (FCS), 2 mM l-glutamine, 50 units/mL peni-
cillin, and 50 ␮g/mL streptomycin. After 24 h at 37 ◦C, nanoparticles
in a similar medium to the cells (100 ␮L) were added, giving total
concentrations of 500, 250, 125, 50, 25 and 12.5 ␮g/mL. After a fur-
ther 24 h at 37 ◦C, 3ꢀ-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-
bis(4-methoxy-6-nitro)benzene-sulphonic acid hydrate (XTT) was
added according to kit manufacturer instructions (Biological Indus-
tries, Beit Haemek, Israel). Absorbance was read at 450 nm,
and the absorbance of corresponding concentrations of PS-FBMS
nanoparticles was subtracted from the reading. Cell viability was
determined as a percentage of the negative control (untreated
cells).
2.7. Uptake of nanoparticles by phagocytes
2.7.1. Cell lines
Cells from the macrophage tumour line RAW264, were grown
as adherent cells in tissue culture dishes in a 5% CO2, 95% air atmo-
sphere at 37 ◦C, similarly to a previous phagocytosis study [25].
Cultures were transferred biweekly in DMEM supplemented with
10% foetal calf serum (FCS), 2 mM l-glutamine, 50 units/mL peni-
cillin, and 50 ␮g/mL streptomycin.
Scheme 1. Synthesis of FBMS.
2.7.2. Endocytosis assay
100 ␮L aliquots of the RAW264 cells (1 × 107 cells/mL DMEM-
FCS) were dispensed into fluorescence activated cell sorter (FACS)
tubes, and 100 ␮L of PS-FBMS beads [1 mg beads/mL DMEM-FCS]
were added to each, and the cultures were incubated at 37 ◦C for
4–5 h. After incubation, PBS (Ca2+, Mg2+ free) was added and the
tubes were centrifuged at 1300 rpm for 10 min. The cells were
resuspended in PBS (1 mL) and fluorescence was determined by
FACS (GalliosTM, Beckman Coulter). In addition, 100 ␮L aliquots
of cell preparations, (1 × 105 cells/mL in DMEM-FCS), were dis-
pensed a 24-well plate and incubated at 37 ◦C. After 24 h, 100 ␮L
of PS-FBMS beads (1 mg beads/mL) were added and the cultures
incubated for 4 h at 37 ◦C. The cells were rinsed with PBS, and
then viewed by a digital inverted fluorescent microscope (EVOS,
Advanced Microscopy Group).
3. Results and discussion
3.1. Fluorescent crosslinker FBMS
The fluorescent crosslinking monomer was synthesised as
described in Section 2, by reaction of fluorescein with p-
chloromethyl styrene, in basic conditions (Scheme 1). The first step
involves deprotonation of fluorescein, opening of the lactone ring
and formation of its dianionic form [14,27] The product was a dark
orange powder (yield 89%).
Interesting to note is that the hydrogen atoms at C23 are
diastereotopic in the NMR spectrum, indicating a biphenyl-like
chirality of the molecule. This is due to substitution at the ortho
position of the aromatic ring (C20) and hence slow rotation at room
temperature about the C13 C15 bond (in the NMR time-scale), due
to steric hindrance.
2.8. Albumin coating
An albumin coating was achieved according to the literature
[26], briefly, a mixture of bovine serum albumin (BSA, 10 mg) and
PS-FBMS nanoparticles (various sizes, 20 mg) in water (10 mL) was
prepared. The mixture was allowed to gradually reach 60◦, and was
then stirred at 60◦ overnight, after which the mixture was allowed
to cool. Excess albumin was removed by cycles of membrane ultra-
filtration, during which the mixture was gradually transferred to
PBS.
Qualitative determination of the presence of albumin on the
particle surface was performed using an immunoperoxidase assay
intended for determination of albumin in human sera (Immunol-
ogy Consultants Laboratory, Inc.). Briefly, duplicate samples of
uncoated as well as BSA coated PS-FBMS nanoparticles (100 ␮L)
were transferred to separate wells containing bound anti-albumin
antibody (antiAlb), and allowed to stand at room temperature
overnight. The wells were then washed to remove unbound par-
ticles, and horseradish peroxidase (HRP) antiAlb conjugate was
added. A solution containing 3,3ꢀ,5,5ꢀ-tetramethylbenzidine and
hydrogen peroxide in citric acid buffer at pH 3.3 was added, fol-
lowed by a 0.3 M sulphuric acid solution, and absorbance was
determined at 450 nm.
3.2. Synthesis of PS-FBMS nanoparticles
Fluorescent PS-FBMS nanoparticles were synthesised as
described in Section 2, by dissolving a small percentage (discussed
in the following sections) of FBMS in styrene, followed by emulsion
polymerisation. Excess reagents (such as unpolymerised monomer
and SDS) were removed by filtration followed by extensive dial-
ysis against water. Variation of concentrations of the fluorescent
crosslinker, and concentrations of the surfactant created a series
of particles with different fluorescence intensities and of different
sizes and size-distributions.
TGA and DSC were performed on the PS-FBMS nanoparti-
cles, and were compared to thermal behaviour of polystyrene.
Polystyrene particles were recorded to undergo thermal decom-
position at 360–440 ◦C, whereas those crosslinked with FBMS
appeared to decompose at 375–455 ◦C, showing increased stabil-
isation against heat, which is probably due to crosslinking of the
polystyrene chains. In addition, the glass transition temperature
(Tg) of polystyrene is at approximately 110 ◦C, whereas the lack of
a Tg indicates the presence of a crosslinked polymer.

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63
Fig. 2. Effect of percentage of FBMS on FI. A linear increase in FI of the particles was
observed with increase in FBMS. Increasing the concentration of particles in water
also increased the fluorescent signal, without quenching.
Fig. 1. Variation of average particle size and size distribution by variation of per-
centage of FBMS, keeping concentrations of initiator, total monomer and volume of
continuous phase constant, as described in Section 2.
3.3. Effects of variation of FBMS concentration
Variations of crosslinker concentrations while preserving the
total monomer concentration may be expected to affect the aver-
age particle size. Solutions of 0.1, 0.5, 0.75 and 1.0% (by weight) of
FBMS in styrene were prepared at room temperature. At 1.0% (by
weight) the solution appears to be saturated, as larger quantities
were not successfully dissolved in the styrene. Following polymeri-
sation, the resulting average particle diameters were 70, 60, 55
and 50 nm respectively (Fig. 1), showing only a slight decrease in
size, and the size-distribution was also not significantly affected.
The trend of a gradual reduction in size is probably due to the
decreased ability of the growing nanoparticle to “swell” during the
emulsion polymerisation process with an increased percentage of
crosslinker.
Fig. 3. Effect of SDS concentration on particle size. A non-linear decrease in particle
size was observed with increasing concentrations of SDS, while initiator concentra-
tion, and volumes of styrene and of aqueous continuous phase are kept constant.
Fluorescence intensity rarely scales linearly with the number of
dye molecules in the particle due to factors such as singlet–singlet
annihilation, self-quenching and Stern–Volmer quenching [1].
However, when the fluorescent moiety is kept at low concentra-
tions relative to the non-fluorescent monomer, when integrated
into a polymer, FI may be increased with an increase in fluorescent
dye, as the non-fluorescent monomer maintains a certain distance
between the fluorescent moieties, thus preventing aggregation and
fluorescence quenching. The quantum yield of FBMS was found to
be 0.67, using fluorescein as a standard with a quantum yield of
0.97 [28]. Despite its lower quantum yield, incorporation of sev-
eral FBMS molecules would result in significant increase in FI per
particle compared to a single fluorescein molecule, if no significant
quenching or non-emissive energy transfer takes place.
approximately 40–100 nm, while keeping constant initiator and
total monomer concentrations. In an experiment using 1% initiator
relative to the monomer, and 15 mL of aqueous continuous phase
with 0.25, 0.5, 0.75, 1, 1.5 and 2% SDS, particles of average diam-
eter, 94, 67, 54, 45, 44 and 37 nm respectively (Fig. 3). PS-FBMS
nanoparticles, at approximate concentrations of 67 mg/mL gave
an orange to yellow suspension, depending on particle size. The
smaller particles gave a darker translucent suspension, whereas
the larger particles gave a lighter opaque suspension (qualitatively
illustrated in Fig. 4).
3.5. Photobleaching
For nanoparticles prepared using 0.1, 0.5, 0.75 and 1.0% (by
weight) of FBMS in styrene, the relative FI was measured for simi-
lar particle concentrations (mg/mL). There appeared to be a linear
increase in intensity with an increase in concentration of FBMS,
with no fluorescence quenching observed (Fig. 2). This can be
explained in that the polystyrene chains provide sufficient sepa-
ration between the fluorescein (FBMS) moieties, so non-emissive
energy transfer cannot take place. In experiments that followed,
FBMS concentrations used were 1.0% (by weight, 0.2 mmol/mol
styrene), in attempt to create the particles with highest FI.
The spectrofluorometer measurements of FI for FITC and for the
PS-FBMS nanoparticles were plotted against time over a twenty-
minute period. While for both FITC and the nanoparticles the
photobleaching appeared to be slow, the decrease in FI was signifi-
cantly slower for the nanoparticles, which over the twenty minutes
3.4. SDS concentration and particle size
As is well documented, particle size decreases with increas-
ing surfactant concentration [21,29–32]. Besides the tightly packed
surfactant molecules affecting growth during particle formation
[21], the smaller the particle, the greater the number of SDS
molecules required per unit of surface area for particle stabilisation
[32]. Variation of SDS concentration was found to be an effective
method for obtaining average particle diameters in the range of
Fig. 4. Appearance of aqueous PS-FBMS nanoparticles (67 mg/mL) of increasing size
(35 5 nm (left) to 87 12 nm (right)).

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M. Pellach et al. / Journal of Photochemistry and Photobiology A: Chemistry 228 (2012) 60–67
Fig. 5. Photobleaching kinetics of 0.01 ␮M aqueous FITC (100 ␮L) compared to
0.2 mg/mL aqueous PS-FBMS nanoparticle suspensions (100 ␮L).
decreased by approximately five percent and more linear compared
to the FITC, which decreased more rapidly by about twenty percent,
and the FI graph gradually plateaus (Fig. 5).
It has been shown that there are two major pathways for
loss of excited-state fluorescein: reaction between a triplet-state
dye molecule with another dye molecule, and reaction between a
triplet dye and an oxygen molecule [16]. In the PS-FBMS nanopar-
ticles, the dye–oxygen interaction is greatly reduced due to the
polystyrene shielding, and dye–dye interactions are practically
eliminated due to the covalent incorporation of the fluorescent
moieties and their lack of mobility. Furthermore, The excitation
wavelength used was the particles’ excitation maximum, rather
than that of aqueous FITC (460 rather than 494). On illumina-
tion at the FITC excitation maximum one would expect a more
rapid decrease in FI, and an even slower loss of fluorescence of the
particles.
Fig. 6. Optical spectra for (a) fluorescein in EtOH. Excitation ꢀmax = 458(485) nm,
emission ꢀmax = 515 nm, (b) FBMS in EtOH. Excitation ꢀmax = 460(488) nm, emis-
sion ꢀmax = 521, and (c) an aqueous suspension of PS-FBMS nanoparticles of average
diameter 50 nm. Excitation ꢀmax = 452 nm, emission ꢀmax = 521.
3.6. Fluorescence spectra
3.6.1. “Solvatochromic” effects
The fluorescence spectra were obtained for fluorescein, the flu-
orescent crosslinker in solution, as well as incorporated within PS
nanoparticles (Fig. 6). Interestingly, when comparing, the spectrum
of fluorescein in EtOH is similar to that of FBMS in EtOH, with
no significant shifts in the excitation and emission maxima. The
optical spectra of fluorescent molecules are often affected by their
medium, as changes in polarity of the medium result in changes in
the electronic state or dipole moments of the fluorescent moieties,
which consequently results in shifts of peak maxima [14,33–35].
Although the FBMS moiety is slightly modified after polymerisa-
tion, a shift is demonstrated with the spectrum of FBMS within
non-polar PS. Note that once dye is incorporated into polymeric
particles, it no longer interacts with the solvent in which the parti-
cles are dispersed, and therefore the media in which the particles
are dispersed does not affect the fluorescence spectra, that is,
solvatochromism is generally not relevant for these polystyrene-
based fluorescent particles, unless the solvent is able to penetrate
them.
Also worth noting, is that the stokes shift of fluorescein in
EtOH, as shown in Fig. 6, is approximately 67 nm, which is advan-
tageous over the usual 22–24 nm observed for aqueous FITC,
however, its use is limited partly because of its lack of solubility
in aqueous solution. The stokes shift of the PS-FBMS nanopar-
ticles is similar to that of fluorescein in EtOH (approximately
70 nm), allowing excitation as well as detection at the parti-
cles’ maxima, and thus full exploitation of their fluorescent
properties, while also having dispersibility in an aqueous
phase.
3.6.2. Particle size and optical effects
The original precipitated product (see Section 2.2) contained
approximately ten percent of the monoalkylated product fluores-
cein methyl styrene (FMS, molecular weight 449 by MS) monomer
(with alkylation presumably at O22, Scheme 1). Following poly-
merisation using this product mixture, the emission spectrum of
the obtained nanoparticles was found to be a shouldered peak (as
in the spectra in Fig. 6), and again approximately equivalent for each
of the particle sizes. The excitation spectra, however, showed a size-
dependence for the PS-FBMS-FMS nanoparticles, with a secondary
excitation peak appearing similar in intensity to the primary peak
for the 100 nm particles, and then decreasing in intensity as particle
size is reduced (Fig. 7). A possible explanation of this phenomenon
is that the monoalkylated fluorescent moiety is less protected by
polystyrene chains. The larger particles’ polystyrene chains are rel-
atively extended, maintaining distance between the fluorescent
moieties. As the particle size decreases, there is increased folding
of the polymeric chains within the particle, allowing an increasing
number of the monosubstituted moieties to reach energy-transfer
distances between fluorescent moieties, and to quench each other’s
fluorescence.

M. Pellach et al. / Journal of Photochemistry and Photobiology A: Chemistry 228 (2012) 60–67
65
Fig. 7. Normalised excitation spectra of PS-FBMS-FMS nanoparticles. Larger particles have a secondary peak appearing similar in intensity to the primary peak, and the
secondary peak decreases in intensity relative to the primary peak, as the particle size decreases.
3.7. Cytotoxicity assay
pH, provides a more “buffer friendly” particle surface, and stabilises
the particles in aqueous buffer solutions such as PBS. The main func-
tion of the proteinaceous coating, though, is to provide functional
groups such as amines and carboxyl groups for conjugation of a
desired targeting agent.
The albumin coating was verified as described in Section 2.
Although the method used was for un-denatured HSA, and the pro-
tein in question was denatured BSA, the method was predicted to
be effective for qualitative analysis, due to sequential and structural
similarities between BSA and HSA [36]. The average absorbance for
uncoated particles at 450 nm was 0.095 (a.u.) compared to 0.145
(a.u.) for coated particles. As expected, the use of denatured BSA
An XTT assay for determining the effect of PS-FBMS nanopar-
ticles on cell viability was performed, as described in Section 2.
After 24 h of exposure of the phagocyte cell line RAW264 to the
nanoparticles, no significant toxicity was observed for nanoparti-
cle concentrations of up to 125 ␮g/mL. Minor toxicity was observed
for concentrations of 250 and 500 ␮g/mL, with approximately 80%
and 75% cell viability, respectively (Fig. 8).
3.8. Uptake of nanoparticles by phagocytes
A macrophage cell line (RAW264) was treated with differ-
ent sized PS-FBMS nanoparticles, as described in Section 2. The
macrophage cells were found to gain fluorescence due to the
uptaken PS-FBMS nanoparticles (Fig. 9). The larger the particles,
the greater the percentage of cells that obtained fluorescence and
the greater their FI (Fig. 10a). A kinetic study showing increas-
ing fluorescence over 5 h indicated continued internalisation of
nanoparticles (Fig. 10b). With illumination of the cells over several
minutes, no loss of fluorescence was observed due to photobleach-
ing.
3.9. Albumin coating
The uncoated particles are stabilised in the aqueous phase due to
the negatively charged SDS molecules that remain on the particle
surface, in slightly acidic aqueous conditions. For most biological
labelling, the particles require a buffered environment, that is, pos-
itive and negative ions that affect both the pH of the solution as well
as the charge of the particle surface, and hence the ability of SDS
molecules to stabilise the particles. Transferring the particles to PBS
thus leads to particle destabilisation and aggregation. The albumin
coating, with both positive and negative charges at physiological
Fig. 9. Fluorescent microscopy of RAW264 macrophages without (a) and containing
(b) uptaken 60 12 nm PS-FBMS nanoparticles. The figure shows that the particles
have been internalised into the cells and are not merely adsorbed onto the cell
surface.
Fig. 8. Viability of cells exposed to PS-FBMS nanoparticles, after 24 h, expressed as
a percentage of the negative control.

66
M. Pellach et al. / Journal of Photochemistry and Photobiology A: Chemistry 228 (2012) 60–67
shift. Each particle also has greater fluorescence and greater pho-
tostability compared to a single FITC molecule.
Functionalisation of the particles is provided by an albumin
coating, which provides both carboxyl and amine groups that may
be used for conjugation of a bioactive agent. In the future, the
bioactivated fluorescent nanoparticles can then be used in vari-
ous biomedical applications such as diagnostics, drug targeting,
cellular labelling and cell separation [3,37]. Overall, the PS-FBMS
nanoparticles are promising fluorescent nanomaterials that are
easily synthesised, bright, photostable and size-tuneable with a
variety of potential uses.
Acknowledgements
Thanks to Dr. Hugo Gottlieb for his assistance in NMR spec-
troscopy analysis and structure elucidation. Also thanks to Dr. Igor
Grinberg for his assistance in performing cytotoxicity assays.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jphotochem.2011.11.012.
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