Three of these copolymers are discussed here: copolymers C1,
C2 and C3, corresponding, respectively to 4.10, 0.85 and
0.16 mol% of RCA in the initial cyanoacetate feed. The
fluorescent copolymers were analysed by 1H NMR spectro-
scopy and showed an excellent correlation with the expected
structure (Fig. S1).w Size exclusion chromatography showed
low number-average molar masses, Mn, with high polydispersity
indexes (Table S1)w due to the significant amount of low-
molar mass amphiphilic oligomers, commonly observed for
this kind of reaction.7,8 No influence of the initial amount of
RCA over the copolymerisation process was observed.
Well-defined nanoparticles were formed by self-assembly in
aqueous medium and characterised by DLS and z-potential
measurements as functions of the RCA initial amount and
time (Table S1 and Fig. S2).w In all cases, stable nanoparticles
were obtained with average diameters in the 115–125 nm range
together with narrow particle size distributions. z-potential
measurements showed negative values from À30.9 to À40.6 mV.
Besides, nanoparticle diameters and surface charge remained
constant over time in aqueous solution at 37 1C, thus
confirming their excellent stability at a temperature relevant
for biomedical assays. Therefore, all these characteristics make
them suitable candidates for drug delivery purposes and cell
imaging.
the nature of the copolymer and associated nanoparticles,
linear evolutions of fluorescence intensity vs. concentration
were observed up to rather high concentrations.
Rhodamine B-tagged PACA nanoparticles were then
employed for in vitro imaging studies on the hCMEC/D3
human brain endothelial cell line, which has been validated
as a unique in vitro model of the human blood–brain barrier
(BBB).9 Prior to imaging studies, cell viability assays
were performed in order to determine the cytotoxicity of
the P(HDCA-co-RCA-co-MePEGCA) nanoparticles on
hCMEC/D3 cells. No statistical difference in cytotoxicity
was observed between nanoparticles containing an increasing
amount of rhodamine B (Fig. S10).w Similarly to non-fluorescent
P(HDCA-co-MePEGCA) nanoparticles, no significant cyto-
toxicity was obtained until a concentration of 30 mg mLÀ1
.
Confocal laser scanning microscopy (CLSM) was then
employed for in vitro imaging studies. Upon microscope
observation, the fluorescent nanoparticles in water appeared
as small, well-defined, fluorescent spots displaying typical
Brownian motion (Fig. S11).w After a 12 h incubation period
of hCMEC/D3 cells with fluorescent nanoparticles (N1), cells
were washed with fresh cell culture medium in order to remove
adsorbed nanoparticles and observed by CLSM. Nomarski
contrast images showed a typical fibroblast shape for the cells
with no morphological alteration, thus supporting the absence
of cytotoxicity as previously shown by cell viability assays (this
observation was made on the basis of numerous images
randomly taken from the cell monolayer). Fluorescence
images superimposed on the Nomarski images showed intense
and fine fluorescence spots accumulated within the cells and
especially around the nuclei (Fig. 2a–c). This observation
suggested that the mechanism by which nanoparticles entered
the cells was governed by endocytosis since fluorescence was
localised into vesicles. However, according to their proximity
to the nuclei, those vesicles are highly supposed to be late
endosomes (Fig. 2d). These results confirmed previous
observations with primary cultures of rat brain endothelial
cells showing that P(HDCA-co-MePEGCA) nanoparticles
were able to penetrate by endocytosis.10
The presence of different amounts of RCA in the copolymers
and associated nanoparticles could be readily perceived under
visible light and UV excitation at 365 nm (Fig. 1). Fluorescent
properties of the materials were then thoroughly studied by
fluorescence spectroscopy (Fig. 1 and S3–S7).w Emission and
excitation wavelengths of the copolymers and nanoparticles
were determined. For instance for copolymer C2, lex = 563 nm
and lem = 583 nm, with a Stokes shift of 20 nm, in good
agreement with the spectral properties of rhodamine B-tertiary
amide derivatives.6 No significant change was observed upon
self-assembly as lex = 568 nm and lem = 583 nm were
recorded for nanoparticles N2. Besides, for a given copolymer
(or nanoparticle) concentration, the fluorescence intensity
decreased when decreasing the initial amount of RCA in the
starting cyanoacetate mixture (Fig. 1).
Eventually, the evolution of the fluorescence intensity of
copolymer solutions and resulting nanoparticle suspensions
was recorded as a function of the concentration, allowing
linear and curved parts to be determined (Fig. S7),w which is
useful for fluorescence intensity-based calculations. Whatever
As long as poly(alkyl cyanoacrylate) nanoparticles are
known to be biodegradable by enzymatic degradation via
hydrolysis of ester functions,2 it was important to assess that
the observed fluorescence intensity was coming from the
fluorescent nanoparticles and not from free rhodamine B
alcohol (released after hydrolysis). After 8 h incubation of
fluorescent nanoparticles at different concentrations with
Fischer rat plasma at 37 1C, only 11–14% of fluorescence loss
was measured (Fig. S12),w in very good agreement with the
in vitro biodegradation profile of non-fluorescent P(HDCA-co-
PEGCA) nanoparticles.11 This result also showed that the
presence of rhodamine B cyanoacetate units in the macro-
molecular structure did not alter the degradation profile of the
nanoparticles.
Under identical experimental conditions and acquisition
settings (detector gain: 535, laser power: 57%), a lower amount
of rhodamine dye covalently linked to the nanoparticles resulted
Fig.
1
Fluorescence emission spectra of P(HDCA-co-RCA-co-
MePEGCA) copolymer solutions in CHCl3 at 0.1 mg mLÀ1 (a) and
of resulting nanoparticle suspensions in water at 0.1 mg mLÀ1 (b).
Inserts: pictures of copolymer solutions (left) and nanoparticle suspensions
(right) under visible light or under UV excitation at 365 nm.
in
a decrease of fluorescence intensity. Indeed, only
faint fluorescent areas were noticed for nanoparticles N3
(Fig. S13).w Nevertheless, by increasing the detector gain up
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2602–2604 | 2603
Brambilla, Davide
