Adapting BODIPYs to singlet oxygen production on silica nanoparticles

Article abstract of DOI:10.1039/c7cp01333g

A modified St?ber method is used to synthesize spherical core-shell silica nanoparticles (NPs) with an external surface functionalized by amino groups and with an average size around 50 nm. Fluorescent dyes and photosensitizers of singlet oxygen were fixed, either separately or conjointly, respectively in the core or in the shell. Rhodamines were encapsulated in the core with relatively high fluorescence quantum yields (Φ_fl ≥ 0.3), allowing fluorescence tracking of the particles. Various photosensitizers of singlet oxygen (PS) were covalenty coupled to the shell, allowing singlet oxygen production. The stability of NP suspensions strongly deteriorated upon grafting the PS, affecting their apparent singlet oxygen quantum yields. Agglomeration of NPs depends both on the type and on the amount of grafted photosensitizer. New, lab-made, halogenated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY) grafted to the NPs achieved higher singlet oxygen quantum yields (Φ_Δ ～ 0.35-0.40) than Rose Bengal (RB) grafted NPs (Φ_Δ ～ 0.10-0.27). Finally, we combined both fluorescence and PS functions in the same NP, namely a rhodamine in the silica core and a BODIPY or RB grafted in the shell, achieving the performance Φ_fl ～ 0.10-0.20, Φ_Δ ～ 0.16-0.25 with a single excitation wavelength. Thus, proper choice of the dyes, of their concentrations inside and on the NPs and the grafting method enables fine-tuning of singlet oxygen production and fluorescence emission.

Full text of DOI:10.1039/c7cp01333g

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Elezcano, R. Prieto-Montero, V. MARTINEZ-MARTINEZ, M. J. J. Ortiz, A. Prieto, E. Pena-Cabrera, J.
Belmonte-Vázquez, I. López-Arbeloa, R. Brown and S. Lacombe, Phys. Chem. Chem. Phys., 2017, DOI:
10.1039/C7CP01333G.
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Darya Radziuk and Helmuth Möhwald
Ultrasonically treated liquid interfaces for progress in cleaning and
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silica core and
performance Φfl
a
BODIPY or RB grafted in the shell, achieving the
0.10-0.20, ΦΔ 0.16-0.25 with a single excitation
~
~
wavelength. Thus, proper choice of the dyes, of their concentrations inside
and on the NPs and the grafting method enables fine-tuning of singlet
oxygen production and fluorescence emission.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Introduction
www.rsc.org/
Production of singlet oxygen (O2(a1
through photosensitization involves three components: a
photosensitizer (PS), light source and oxygen. The
∆
ꢀ), 1O2 in the following)
Adapting BODIPYs to singlet oxygen
production on silica nanoparticles
a
development of photosensitizing materials is an active
research field since singlet oxygen is very useful for numerous
applications. Industrial uses include oxidation reactions of high
added value under mild conditions such as oxidation of fine
chemicals in organic solvents1,2 and wastewater treatment.3
Clinical uses include bactericidal or cancer treatments by
applying photodynamic therapy.4,5,6 In all applications, the
photosensitizer has to be deposited, embedded or grafted on
an inert substrate with suitable form, specific surface area and
surface properties.7 Such supported PSs have numerous
advantages: improved recovery of unspent materials, control
of aggregation and self-quenching of the PS, increase of PS
photostability, possible use of solvents where the PS is poorly
soluble or even use in solvent-free reactions.
Nerea Epelde-Elezcano,a Ruth Prieto-Montero,a Virginia
Martínez-Martínez,a* María J. Ortiz,a Alejandro Prieto-
Castañeda,b Eduardo Peña-Cabrera,c José L. Belmonte-
Vázquez,c Iñigo López-Arbeloa,a Ross Brownd and Sylvie
Lacombed*
A modified Stöber method is used to synthesize spherical core-shell silica
nanoparticles (NPs) with an external surface functionalized by amino groups
and with an average size around 50 nm. Fluorescent dyes and
photosensitizers of singlet oxygen were fixed, either separately or
conjointly, respectively in the core or in the shell. Rhodamines were
encapsulated in the core with relatively high fluorescence quantum yields
Tailor-made nanoparticles (NP) used in the field of cancer
therapy and diagnosis, are of current interest to enhance
activity, delivery and targeting.8,9 However several problems
may arise such as the stability of the NP suspensions in various
solvents due to aggregation or interaction between dyes in the
same particle. Here, we focus on these aspects for silica NPs.
Indeed, among NPs, core-shell silica nanoparticles provide an
opportunity to introduce different types of compounds with
various properties, either grafted on the functionalized NP
surface (shell) or embedded inside pores (core). Therefore, an
increasing number of researchers have considered the use of
(Φfl
≥ 0.3), allowing fluorescence tracking of the particles. Various
photosensitizers of singlet oxygen (PS) were covalenty coupled to the shell,
allowing singlet oxygen production. The stability of NP suspensions strongly
deteriorated upon grafting the PS, affecting their apparent singlet oxygen
quantum yields. Agglomeration of NPs depends both on the type and on the
amount of grafted photosensitizer. New, lab-made, halogenated 4,4-
difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY) grafted to the NPs
achieved higher singlet oxygen quantum yields (ΦΔ ~ 0.35-0.40) than Rose
Bengal (RB) grafted NPs (ΦΔ ~ 0.10-0.27). Finally, we combined both
fluorescence and PS functions in the same NP, namely a rhodamine in the
silica nanoparticles for numerous medical applications10–12
:
platforms for PDT,13–16 bioimaging probes,17–19 combined uses
such as theragnostics (therapy and diagnostics),20–22 or drug
delivery.23,24 The main advantages of silica NPs are chemical
inertness, transparency, porosity tuning insensitive to swelling
or alterations with pH, good biocompatibility and easy
functionalization of their surface.25,26 A silica matrix thus
provides a stable chemical and mechanical environment for an
encapsulated fluorophore, protecting it from external
perturbations,27 which is of special interest in bioimaging
where NPs can be monitored by optical techniques both in
vitro and in vivo.28 Conversely, the PS is better grafted on the
outer surface to favour sensitization and delivery of singlet
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oxygen. This implies that a functional group on the PS reacts intersystem crossing to the triplet state.5,32,34,35,36,37 BODIPYs
with another functional group on the silica substrate. Among can also be modified to introduce grafting functionality to
classical PSs, such as xanthene dyes, anthracene derivatives, various solid substrates via carboxylate or amino groups.
porphyrins and phthalocyanins or aromatic ketones,29,7 the In this work, we compare Rose Bengal and two new lab-made
carboxylate moiety of commercial Rose Bengal is easily BODIPYs when grafted to core-shell silica NPs, particularly
coupled to various substrates.30,31 Rose Bengal is a popular singlet oxygen quantum yields (ΦΔ) and NP aggregation due to
reference for singlet oxygen production in polar solvents such surface modification. The coupling process was first optimized
as in ACN or MeOH because of its high singlet oxygen quantum with commercial Rose Bengal (RB1) and further applied to the
yield (ΦΔ 0.5-0.8 in the visible region,
λ
abs 550 nm). Its principal BODIPYs (Figure 1) since to the best of our knowledge the
grafting of BODIPYs on nanoparticles for singlet oxygen
disadvantage is its susceptibility to photobleaching.
On the contrary, BODIPYs (base motif 4,4-difluoro-4-bora- generation was up to now hardly investigated.38,39 In a second
3a,4a-diaza-s-indacene) are better known for their step, we describe core-shell NPs with dual activity, combining
photostability and high extinction coefficients in the visible both imaging, by embedding fluorescent rhodamines in the
region than for the sensitization of singlet oxygen.32,33 Here we core, and singlet oxygen production, by grafting singlet oxygen
exploit the versatility of BODIPY synthesis to produce a novel photosensitizers in the shell. We also show the importance of
graftable sensitizer of singlet oxygen. Indeed, although BODIPY controlling Förster resonance energy transfer (FRET) in such
dyes usually exhibit negligible efficiency of triplet formation small particles. Combining two dyes in one particle can then
and high fluorescence quantum yields, the incorporation of achieve the contradictory aims of high fluorescence yield and
heavy atoms (mainly iodine) directly to the BODIPY core high singlet oxygen sensitization with a single excitation
enhances the spin–orbit coupling, thereby favouring wavelength.
Figure 1. Structure of the used dyes
Results and discussion
In this work, mesoporous silica nanoparticles were prepared
according to the Stöber methodology based on the sol-gel
process with an additional reactant added to the system,
namely a surfactant:40,41 in the first step the core was
synthesized through the mixture of an alkoxide in
a
water/alcohol solvent using ammonium hydroxide as a catalyst
and CTAB (cetyl trimethylammonium bromide) surfactant. In a
second step, addition of 3-aminopropyltrimethoxy-silane
(APTMS) builds the shell containing amino groups for surface
functionalization (Figure 2).
Figure 2. Synthesis of the core-shell silica nanoparticles by modification of the Stöber
method.
For imaging applications, two highly fluorescent rhodamines was achieved via Suzuki coupling of p-carboxyphenylboronic
(Rh6G, λem 530 nm and Rh640, λem 550 nm, Figure 1) were acid with thioBODIPY followed by iodination to yield the same
introduced in the initial alcohol solvent of the core xerogel. compound as in 43. BDP2 was prepared in two steps by
The PS grafting process at the surface (shell) was first iodination of the thioBODIPY, followed by nucleophilic
optimized with Rose Bengal (RB1 and RB2) and implemented substitution of the thiomethyl group with (3-
with alternative lab-made BODIPYs (BDP1 and BDP2) (Figure aminopropyl)triethoxysilane. Two grafting methods were
1). Original synthesis schemes were used, based on the evaluated: either a peptide coupling reaction between surface-
starting thioBODIPY, 3,5-dimethyl-8-methylthioBODIPY.42 The NH2 of the silica shell and PS containing a carboxylic group
synthesis of BDP1 with a carboxyphenyl group in meso position such as RB1 or BDP1 (NP-RB1, NP-BDP1) or direct
2 | J. Name., 2012, 00, 1-3
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polymerization of a PS containing a triethoxysilyl group (RB2 or
BDP2) on the surface OH-group of silica (NP-RB2 or NP-BDP2)
(Scheme S1, ESI).
Table 1. Spectrophotometric estimation of the amount of grafted PS on silica NPs
(suspensions in CHCl3) and % atomic concentration on their surface from XPS results.
Sample
% At Concentration
Si Cl*
Meth.1‡ Meth.2‡ 15 52 4.0 26 2.5
PS amount
(ꢀ
mol gr-1)
C
O
N
F
-
I
NP
-
3.1. Characterization and optimization of the silica
nanoparticles
NP-BDP1
NP-BDP2
NP-RB1
NP-RB2
-
-
1.8
5.2
-
22 44
7
22
2.3 2.2
23 42 5.1 24 2.4 2.2 1.6
8.0
24
-
-
-
-
-
-
-
-
-
-
-
-
-
-
The composition of the NPs was derived from XPS results
(Table 1). XPS data confirmed the major contribution of SiO2
for NPs and the relative atomic ratio of N atoms around 4 % in
the bare particles. The presence of the I and F atoms, as well
as the higher contribution of the C and N atoms on both NP-
BDP1 and NP-BDP2, are indicative of the successful grafting of
BDP.
-
‡ More details about method 1 and method 2 in Experimental Section.
* The presence of chlorine atoms in bare NP and NP-BDP2 (2.5 % atomic ratio) is due to
the washing of the nanoparticles by a diluted hydrochloric solution at the end of the
synthesis, transforming the amino groups into ammonium chloride groups. For NP-RB1
and NP-BDP1, chlorine atoms were removed by addition of triethylamine.
IR-ATR of the grafted NP-RB1, NP-RB2 and NP-BDP1 confirmed
the presence of amide I (around 1630 cm-1, C=O stretch) and
amide II bonds (around 1550 cm-1, C-N stretch and N-H bend)
(Figure S2, ESI).44 Conversely, this amide bond is not present in
NP-BDP2 since they are obtained by direct polymerization of
BDP2 on OH groups of the silica surface.
All samples analyzed at t0 showed only one distribution curve
with similar hydrodynamic diameters (212±13 nm and 257±54
nm in ethanol and water respectively, indicating
agglomeration of the NPs in these solvents. However, in ACN,
the NP size distribution was considerably lower showing a
better dispersion of NPs (92±3 nm). Nevertheless, important
changes in the hydrodynamic diameter occurred after 1 hour
without stirring; in ethanol higher aggregates were formed,
while in water and ACN two different NP populations were
obtained with larger aggregates together with better dispersed
NPs (Table S1 and Figure S1, ESI). According to the DLS
experiments, ACN was an appropriate solvent for the bare
silica nanoparticles. The DLS results for the different grafted-
NP in ACN and in CHCl3 just after magnetic stirring (t0) are
included in Table S2. After the grafting process, depending
both on the type of photosensitizer and on the grafting
method, the stability of the suspension is greatly modified, as
shown by the increase of the hydrodynamic diameter in ACN,
in the order bare NP< NP-BDP1<NP-BDP2<NP-RB1. Generally
speaking, BDP-grafted NPs are more stable in suspensions
compared to RB-grafted NPs.
According to Table 1 (more details in Experimental Section),
the amount of PS grafted on the silanol groups (NP-RB2 and
NP-BDP2) is slightly higher than the PS grafted on the
functional NH2 groups (NP-RB1 and NP-BDP1). This reflects the
higher amount of silanol groups compared to active NH2
groups.
The nanoparticles had diameters around 50 nm,
homogeneously distributed according to the SEM image
(Figure 3 left) with a shell thickness around 10 nm (Figure 3
middle). The mesoporous structure of the silica NP is also
nicely observed by TEM (Figure 3 right). TEM images also
confirmed that the grafting process did not modify the
spherical morphology and distribution, with particles size
around 50 nm before and after grafting (Figure S3). Dynamic
light scattering (DLS) measurements were performed to
evaluate the NP size distribution, their dispersion and stability
in different solvents with different polarity such as water,
ethanol and acetonitrile (Table S1, Figure S1, ESI). The DLS
measurements were done just after magnetic stirring of the
NPs in a given solvent (t0) and after one hour (t1) without
stirring.
Figure 3. (Left) SEM images of silica NPs with around 50 nm size; (Middle) TEM image of the Core-Shell Silica NP and (Right) TEM image of the mesoporous structure of the silica
NPs.
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consequence of the better stability of the chloroform
suspension compared to NP-RB2 (singlet oxygen values lower
than 0.10). These results emphasize the importance of
controlling the agglomeration of the NP-grafted
photosensitizers. It is thus of crucial importance to obtain
stable nanoparticle suspensions to obtain good singlet oxygen
production for future implementation.
3.2. Photophysical properties and singlet oxygen
production by PS grafted on NPs
Contrary to bare NPs, no suitable solvent was found to obtain
stable nanoparticle suspension for NP-RB2, while in the less
polar chloroform, NP-RB1 suspension was more stable (Figure
S3, ESI).45 We refer here to resistance against precipitation not
photostability. On the contrary, stable suspensions of NP-BDP1
and NP-BDP2 were obtained in both acetonitrile and
chloroform. Contrary to bare NPs, fast precipitation of both
NPs was observed in water. These data suggest significant
surface modification on NP-RB samples, while surface
modification was much less significant on NP-BDP.
Figure 4. (A) Absorption spectra in ACN of NP-RB1 (red) and NP-RB2 (green) and
of RB in diluted solution (black), (B) Absorption spectra in CHCl3 of NP-RB1 (red)
and NP-RB2 (green) (the spectra of RB in ACN solution (black) is also inserted for
comparison), (C) Absorption spectra of BDP1 solution in ACN (black), NP-BDP1
suspension in ACN (red), NP-BDP1 suspension in CHCl3 (green); (D) Absorption
spectra of NP-BDP2 in ACN (red), in CHCl3 (green) and of BDP2 in THF solution
(black).
All the absorption bands of NP-grafted PSs are slightly broader
than in solution (Figure 4).44,46 The absorption spectra of NP-
RB1 and NP–RB2 show a main band centred at 561 nm with a
shoulder around 514 nm, more prominent than in solution
(Figure 4 A and B). On the other hand, the absorption spectra
of NP-BDP1 and NP-BDP2 show no significant modification in
any solvents (Figure 4 C and D).
B
A
At first sight, these observations are consistent with the well-
known tendency of xanthene dyes (RB) to aggregate in
concentrated or rigid media. Indeed this kind of spectral
deformation is often interpreted as an indication of e.g. H-
aggregation.29,44,47,48 Indeed, assuming purely random local
fluctuations of the inter-dye molecule distances, from point to
point on a NP, the classical Hertz distribution law in two
dimensions implies significant fractions of paired molecules
(see detailed calculations in ESI, §S4, Eqs S1-S5). However one
must beware of artefacts in optically thick samples. This type
of spectral shape, where a “new” shoulder is observed close in
500
600
500
600
wavelength(nm)
wavelength(nm)
D
C
wavelength to an existing weak vibronic band is discussed in
49
detail in
and references therein. The observed spectral
500
600
400
500
changes could be due to the influence of multiple scattering in
the sample. The fact that these scattering effects are much less
severe in NP-BDP samples is in agreement with their already
mentioned more stable suspensions (Table S2, Figure S4) and
suggests that dye-mediated NP surface modification controls
particle aggregation, hence scattering and the effective optical
path length and finally the shape of the spectra. Unfortunately
the small amounts of available NPs precluded further
investigating these effects.
wavelength(nm)
wavelength(nm)
For the same reason, higher singlet oxygen quantum yields
were obtained with NP-BDP, which remain stable in ACN and
CHCl3 suspensions (0.33 and 0.37 for NP-BDP1, 0.36 and 0.38
for NP-BDP2, respectively, Figure S5). These results are in
agreement with the absorption spectra, indicating that both
NP-BDPs are much less agglomerated. Thus, by using these
BODIPYs as grafted PS, the range of solvent polarities suitable
for stable NP suspensions is wider than with RB, leading to
higher singlet oxygen quantum yields (although lower than
with the corresponding BDP in solution, Table 2).35 These
results are in agreement with previous data on singlet oxygen
detection with NPs containing a covalently encapsulated
Note here that in the case of NP-RB, the dye-mediated
agglomeration of NPs not only modifies the absorption spectra
but also explains the reduced singlet oxygen generation (Table
2). Nonetheless, a higher singlet oxygen quantum yield was
obtained with NP-RB1 in CHCl3 suspension
(
S5) relative to ACN Φꢀ= 0.14, Figure S5)
Φꢀ= 0.27, Figure
likely as a
(
,
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BODIPY similar to BDP1 (with four methyl groups on the detection under conventional fluorescence microscopes and
indacene core) in toluene suspension. However no singlet therefore adequate for bio-imaging (Figure S7 D and E).
oxygen quantum yield was measured in this earlier study.50 Two different strategies were designed to combine the
Triplet quantum yields would in general be a very useful fluorescent dye with the photosensitizer in the same NP: i)
complement to the singlet quantum yields but was not different absorbance range (NP noted NP/Rh640-BDP2) in
achievable within the scope of this paper or, apparently, in the order to selectively activate the different actions (fluorescence
literature on this kind of systems. It is to be hoped that future under green excitation and singlet oxygen production under
work in this field will address this point. 35,38,50
blue light) by using different wavelengths; ii) similar absorption
range for both dyes (NP noted NP/Rh6G-RB1 and NP/Rh640-
BDP1) in order to use only one excitation light to trigger both
functions (Figure 5).
Table 2. Photophysical data and quantum yield of singlet oxygen production
of grafted nanoparticles compared with the corresponding dyes in ACN or
CHCl3 solutions and of dual NP/R640-BDP1 and NP/R6G-RB1 suspended in
O
RB1
CHCl3.
NH
Rh640
O Si
BDP2
Rh6G
ΦΔ
O
λabs (nm)
555
λfl (nm)
579
Φfl
ACN
CHCl3
NP/Rh640ꢁBDP2
NP/Rh6GꢁRB
RB1
0.12
0.521
-
BDP1
0.142
0.102
0.662
0.332
0.781
0.361
-
0.273
N
H
NP-RB1
NP-RB2
561
561
550
560
426
430
568
530
564
561
580
584
578
580
484
494
590
550
583
580
0.08
0.03
0.03
≤0.01
0.02
≤0.01
0.56
0.30
0.10
0.20
Rh640
0.073
NP/Rh640ꢁBDP1
BDP1
-
0.373
-
Figure 5. Scheme of the various NPs with dual functionalities.
NP-BDP1
BDP2
The absorption spectra of NP/Rh640-BDP2 in chloroform
suspension (Figure 6) show the characteristic bands of both
dyes at 438 nm (BDP2) and 572 nm (Rh640). The BDP2
absorption band is around 10 times more intense than that of
Rh640, indicating a higher amount of the photosensitizer on
the surface with respect to the fluorescent dye in the core, in
agreement with the estimated concentration for each dye (5.2
NP-BDP2
NP-Rh640
NP-Rh6G
NP/Rh640-BDP1
NP/Rh6G-RB1
0.383
-
-
-
-
0.163
and 0.67 ꢂ
mol g-1, Table 1 and Table S3). The emission band of
-
0.253
Rh640 at 590 nm is also observed upon direct excitation of
BDP2 (λex = 440 nm) where the absorbance of the Rh640 is
practically negligible. In line with this observation, the
contribution of the BDP2 chromophore is also clearly seen in
the excitation spectrum of NP/Rh640-BDP2, at the emission
wavelength of Rh640 (575 nm) (Figure 6C). Moreover, the
fluorescence decay of BDP2 is faster in presence of Rh640
(Figure 6D). All these data confirm that the excited state of
1measured in our laboratory by direct method with phenalenone as reference (Φꢀ = 1
in ACN) 51
2Reference RB in ACN (Φꢀ=0.52)
3Reference 8-methylthio-2,6-diiodoBODIPY, 2I-ThioBDP in CHCl3 (Φꢀ = 0.85).35
3.3. NPs with dual functionality
With a view to tracing the particles (environmental tracing, Rh640 is populated by a Förster Resonance Energy Transfer
recovery or bio-imaging), a fluorescent dye (Rh6G or Rh640) process (FRET) from BDP2 to Rh640. The FRET is
a
was embedded in the silica nanoparticle. In order to optimize consequence of the overlapping between the fluorescence
the fluorescent properties of the rhodamine core, different band of BDP2 (donor) and the absorption band of Rh640
concentrations of Rh6G in the alcohol of the sol-gel synthesis (acceptor) and its efficiency depends on the distance between
were checked in the range of 10-2 - 5.0 x 10-5 M. The dye them, which is typically in the range of 1-10 nm.53 Indeed, the
encapsulation did not modify the NP size, morphology and thickness of the shell, around 10 nm, allows FRET process from
polydispersity (Figure S6, ESI). According to the photophysical the BDP2 at the external surface to the fluorescent dye in the
properties of the fluorescent nanoparticles thus obtained core of the silica nanoparticles. However, the FRET induces a
(Table S3, Figure S7 and Figure S8 in ESI), the optimum negative consequence in the singlet oxygen quantum yield of
concentration of rhodamines in the gel was about 5 x 10-4 M, NP/Rh640-BDP2: it is practically negligible due to a reduced
achieving a dye loading in the core around 1 µmol g-1 (Table intersystem crossing process from the singlet excited state of
S3). As already reported, the fluorescence emission was higher BDP2 to its triplet state in favour of energy transfer process
at low dye loading.22,52 More importantly, the fluorescence from the singlet excited state of BDP2 to the singlet excited
quantum yields of the powders (Φflu = 0.30 and 0.52 for Rh6G- state of R640.
NP and Rh640-NP, respectively), although lower than that of However, when combining the photosensitizer and the
the dyes in solution (Table S3 in ESI), are high enough for fluorescent dye with similar adsorption range in NP/Rh640-
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BDP1 and NP/Rh6G-RB1, individual contributions in the (1.8 µ
mol g-1). On the other hand, for NP/Rh6G-RB1, the
absorption spectra are poorly distinguished. In the case of contribution of Rh6G appears in the stronger shoulder at 520
NP/Rh640-BDP1, the maximum absorption falls between those nm compared to NP/RB1 (Figure 7B). The estimated amont of
of both dyes (Figure 7 and Table S2), indicative of the presence Rh6G (0.67 µ
mol g-1, Table S2) is about 5 times less than RB1
of both chromophores, Rh640 (1.0
µ
mol g-1) and BDP1 (3.3
µ
mol g-1, Table 1) grafted on the external surface.
104
A
B
C
D
103
102
101
100
300
400
500
600
500
600
700
400
500
600
0
10
20
30
wavelength (nm)
wavelength (nm)
wavelength (nm)
Time (ns)
Figure 6. (A) Absorption spectra and (B) fluorescence spectra of NP/Rh640-BDP2 with λex 440 nm (black) in CHCl3. BDP2 (blue dotted lines) and Rh640 (red dotted lines) in a EtOH
are inserted; (C) excitation spectra of NP/Rh640-BDP2 suspension in CHCl3 at λem 575 nm (black), and the absorption spectra of BDP2 in ACN solution (blue dotted line) and Rh640
in EtOH solution (red dotted line). D) Fluorescence decay curves of NP/BDP2 (blue) and NP/Rh640-BDP2 (black) upon 400nm and 490 nm emission wavelength.
Importantly, NP/Rh640-BDP1 and NP/Rh6G-RB1 achieve a the core (fluorescein isothiocynate, FITC) and
a PS
balance between the singlet oxygen quantum yield and the (hematoporphyrin, HP) in the shell.54 In their case, the dual
fluorescence quantum yields (Table 2). Particularly, NP/Rh640- functionality in water suspensions was obtained using
BDP1 has a fluorescence quantum yield around 0.10 (Φfl = 0.56 different triggering excitation light: 488 nm for fluorescence
for NP-Rh640) together with a singlet oxygen quantum yield of emission and 633 nm for singlet oxygen production. Estevao et
0.16 (ΦΔ = 0.37 for NP-BDP1). Even better, NP/Rh6G-RB1 has a al., obtained dual functionality in aqueous suspensions of core-
fluorescence quantum yield around 0.20 (Φfl = 0.30 for NP- shell nanoparticles containing fluorescent rhodamine B in the
Rh6G) with a relatively high singlet oxygen quantum yield of core and verteporphin in the shell as PS for singlet oxygen
0.25 (ΦΔ = 0.27 for NP-RB1).
production, triggered by two distinct excitation wavelength
(520 and 690 nm respectively).22
Experimental
2.1. Synthesis of BDPs
Materials and methods
All starting materials and reagents were commercially
obtained, unless indicated otherwise, and used without
further purifications. Common solvents were dried and
distilled by standard procedures. Flash chromatography was
performed using silica gel (230-400 mesh). NMR spectra were
recorded at 20 ºC in CDCl3. 1H chemical shifts are dated in ppm
Figure 7. (A) Absorption spectra in CHCl3 of; (A) NP/Rh640-BDP1 (red), NP-BDP1 (black)
and NP-Rh640 (green), and (B) NP/Rh6G-RB1 (red), NP-Rh6G (black) and NP-RB1
(green).
relative to tetramethylsilane (
standards. 13C chemical shifts are dated in ppm with CDCl3 (
δ = 0.00 ppm) as internal
δ
=
Therefore, the combination of two dyes with complementary
actions (high fluorescence vs high singlet oxygen production)
but with overlapping absorption bands produced samples with
dual functionality, which could be interesting for bio-imaging
and PDT. Other combinations of chromophores can be
combined with the aim of optimizing the dual action of NPs.
The difficulty is reconciling the dual activity with the
constraints of energy transfer. Earlier solutions to this problem
required two excitation wavelengths. Zhang et al. designed
dual core-shell nanoparticles embedding a fluorescent dye in
77.03 ppm) as the internal standard. DEPT 135 experiment was
used to assign the type of carbon nucleus (C vs CH vs CH2 vs
CH3). FTIR spectra were obtained from neat samples using the
ATR technique. High-resolution mass spectrometry (HRMS)
was performed using the EI technique. All the chemicals were
purchased from Sigma-Aldrich, except BDP1 and BDP2 whose
synthesis is show in Scheme 1. Detailed experimental
procedures are given below. See the Supporting Information
for the NMR spectra.
6 | J. Name., 2012, 00, 1-3
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ARTICLE
Synthesis of BDP1
Synthesis of BDP-COOH. Schlenk tube equipped with a stir bar
was loaded with 3,5-dimethyl-8-methylthioBODIPY (100.0 mg,
0.38 mmol, 1.0 equiv.), p-carboxyphenylboronic acid (187.1
mg, 1.13 mmol, 3.0 equiv.), Pd2(dba)3 (8,6 mg, 0.009 mmol, 2.5
mol%), tri-2-furylphophine (6.5 mg, 0.028 mmol, 7.5 mol%),
CuTc (214.2 mg, 1.13 mmol, 3.0 equiv.) and dry THF (0.03 M).
The mixture was bubbled with N2 for 5 min. The Schlenk tube
was then immersed in a preheated oil bath at 55 ºC. The oil
bath was removed after the starting material was consumed
(90 min). After the mixture reached room temperature, the
crude material was absorbed in silica gel, the solvent
evaporated in vacuo, and the product was purified by flash
chromatography using 20% EtOAc/hexanes as eluent. The
desired product was obtained as a dark red solid (77.6 mg,
CO2H
CO2H
ii
I
I
N
N
N
N
B
F
B
F
F
F
BDP-COOH (61%)
BDP1 (25%)
i
SMe
SMe
1
0.228 mmol, 61%). H NMR (500 MHz, CDCl3) δ 8.22 (d, J = 7.5
ii
Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H), 6.66 (s, 2H), 6.24 (d, J = 3.4 Hz,
2H), 2.66 (s, 6H) ppm.43
I
I
N
N
N
N
B
B
Synthesis of BDP1. To a solution of 8-p-carboxyphenyl-3,5-
dimethylBODIPY (BDP-COOH) (45.4 mg, 0.13 mmol, 1.0 equiv.)
in acetic acid (6 mL) was added dropwise a solution of N-
chlorosuccinimide (48.0 mg, 0.36 mmol, 3.0 equiv) and NaI (54
mg, 0.36 mmol, 3.0 equiv.) in 4 mL of acetic acid. The reaction
mixture was stirred at room temperature and monitored by
TLC (EtOAc:hexane:acetic acid (15:84.5:0.5)). The reaction was
stirred for 45 minutes. The crude material was neutralized with
a saturated solution of NaHCO3 in water and the product was
extracted with CH2Cl2 (2 x 50 mL) and washed with water (2 x
25 mL). The combined organic layers were washed with brine
(2 x 100 mL), dried over MgSO4, filtered, and the volatiles were
removed in vacuo and the product was purified by flash
chromatography on silica gel using EtOAc/hexanes/acetic acid
(30:69:1) as eluent. The desired product was obtained as a red
waxy solid (0.02 mg, 0.034 mmol, 25%). 1H NMR (500 MHz,
DMSO-d6) δ 8.09 (d, J = 7.4 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H),
7.09 (s, 2H), 2.56 (s, 6H) ppm.30
F
F
F
F
2I-ThioBDP (94%)
iii
HN
SiOEt3
I
I
N
N
B
F
F
BDP2 (53%)
Scheme 1. Synthesis of BODIPYs BDP1 and BDP2
Synthesis of BDP2. To a solution of 2I-ThioBDP (58.6 mg, 0.11
mmol, 1.0 equiv.) in dry CH3CN (5 mL) and CH2Cl2 (5 mL) was
added dropwise (3-aminopropyl)triethoxysilane (0.04 mL, 0.17
mmol, 1.5 equiv) under argon atmosphere at room
temperature and the resulting mixture was stirred for 1 h. The
solvent was removed in vacuo and the crude product was
dissolved in CH2Cl2, washed with 10% aqueous HCl solution
and water, dried over MgSO4, filtered, and the volatiles were
removed in vacuo and the product was purified by flash
chromatography on silica gel using hexane/CH2Cl2 (5:5) as
eluent. The desired product was obtained as a yellow solid
Synthesis of BDP2:
Synthesis of 2,6-diiodo-3,5-dimethyl-8-methylthioBODIPY (2I-
ThioBDP). To a solution of 3,5-dimethyl-8-methylthioBODIPY42
(32.0 mg, 0.12 mmol, 1.0 equiv.) in acetic acid (2 mL) was
added dropwise a solution of N-chlorosuccinimide (48.0 mg,
0.36 mmol, 3.0 equiv) and NaI (54 mg, 0.36 mmol, 3.0 equiv.)
in 2 mL of acetic acid. The reaction mixture was stirred at room
temperature and monitored by TLC (EtOAc:hexane: (15:85)).
The reaction was stirred for 30 minutes. The crude material
was neutralized with a saturated solution of NaHCO3 in water
and the product was extracted with ethyl ether (3 x 15 mL) and
washed with water (4 x 10 mL). The combined organic layers
were washed with brine (2 x 50 mL), dried over MgSO4,
filtered, and the volatiles were removed in vacuo and the
product was purified by flash chromatography on silica gel
using EtOAc/hexane (2:8) as eluent. The desired product was
obtained as green crystals (58.6 mg, 0.113 mmol, 94%). 1H
NMR (300 MHz, CDCl3) δ 7.50 (s, 2H), 2.76 (s, 3H), 2.61 (s, 6H)
ppm.).55
1
(41.4 mg, 0.06 mmol, 53%). M.p. 130.8-131.6 ºC. H NMR (700
MHz, CDCl3) δ 7.13 (broad s, 2H, 2CH), 6.93 (t, J = 4.2 Hz, 1H ,
NH), 3.88 (q, J = 7.0 Hz, 6H, 3CH2), 3.62 (q, J = 7.0 Hz, 2H,
NCH2), 2.56 (s, 6H, 2CH3), 1.97 (quint, J = 7.0 Hz, 2H, CH2), 1.26
(t, J = 7.0 Hz, 9H, 3CH3), 0.79 (t, J = 7.0 Hz, 2H, SiCH2) ppm. 13
C
NMR (176 MHz, CDCl3) δ 148.4, 129.7, 124.2 (CH), 121.1, 71.8
(C-I), 58.9 (CH2), 49.3 (CH2), 22.1 (CH2), 18.3 (CH3), 14.5 (CH3),
7.9 (CH2) ppm. FTIR ν 3309, 2922, 2853, 1719, 1583, 1556,
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Journal Name
1465, 1369, 1282, 1164, 1080, 960, 794 cm-1. HRMS m/z argon, APTMS (2.8 x 10-2 mmol) was added to get RB2 within
691.0203 (calcd for C20H30BF2I2N3O3Si: 691.0207).
2.2.Synthesis of the core-shell nanoparticles
30 minutes. Immediately the NPs (15 mg) were added and the
reaction mixture was stirred for 3 h. The grafted NP-RB2 were
washed with THF until the surpernatant was completely
colorless and collected by filtration, (Scheme S1, ESI).
First, 0.1 g of CTAB was dissolved in 50 mL of NH4OH at 60 ºC.
When CTAB was dissolved, 0.8 mL TEOS (0.2 M in EtOH) was
added. After 5 h with vigorous stirring at 60 ºC, 0.8 mL of TEOS
(1 M in EtOH, 0.8 mmol) and 0.8 mL of an APTMS solution
(12% v/v in EtOH, 0.007 mmol) were added and left 24 h more
at 60 ºC (Figure 2).
For bioimaging, Rhodamine dyes (10-2 to 5.0 x10-5 M) were
directly added to the synthesis gel. After 24 h, the temperature
was decreased to 20 ºC and the mixture was left with vigorous
2.5. Grafting of BDP2 on surface silanols (NP-BDP2)
BDP2 (5.1 x 10-2 mmol) was dissolved in dry THF (20 mL) under
argon. Then NPs (28 mg) were added and the reaction mixture
was stirred for 3 h. Immediately the NPs were washed with
THF until the surpernatant was completely clean and collected
by filtration (Scheme S1, ESI). For NPs with dual properties,
the synthesis procedures described above were combined
(embedding rhodamine in the core and grafting different PS in
the shell).
stirring for 12
h more. The NPs were collected by
centrifugation of the NP suspension at 12000 rpm at room
temperature for 10 min. The collected solid was washed three
times in a mixture of miliQ water/EtOH and a fourth time with
EtOH only. The surfactant was removed by stirring the NPs
with concentrated HCl (0.2 g of HCl in EtOH) for 12 h. The NPs
were collected by filtration.
2.6. Characterization
The nanoparticles will be denoted NP (without embedded
The size, shape and morphology of the silica nanoparticles
were characterized by scanning electron microscopy (SEM) and
transmission electron microscopy (TEM). SEM images were
recorded on a JEOL JSM-6400 and TEM images were produced
on a Philips SuperTwin CM200 at 200 kV. The nanoparticle size
distribution was analyzed with ImageJ software. Dynamic light
scattering (DLS) was used to estimate the NP size in suspension
(Malvem Zetasizer Nano ZS with Helium-Neon (λ = 633 nm)
laser.
Rhodamine), NP-Rh6G-x (with x the number of
ꢀmole of
Rh6G/g of xerogel) or NP-Rh640 depending on the Rhodamine
used. The amount of dye embedded into the NP “core” was
estimated photometrically, by dissolving the silica matrix with
KOH.
2.3. Grafting of photosensitizers on the NP surface
through NH2 groups (NP-RB1 and NP-BDP1)
The elements at the sub-surface of the mesoporous silica
RB1 (3 x 10-2 mmol) was first dissolved in dry THF (20 mL). nanoparticles were analysed by X-Ray photoelectron
Then N-hydroxysuccimide (NHS, 0.49 mmol) and N-(3- Spectroscopy (XPS, SPECS equipment). The measurements
(dimehylaminopropyl)-N’-ethylcarbodiimide (EDC, 0.46 mmol) were carried out by wide scan: energy step 0.1 eV, dwell time
was added to activate the carboxylic groups of the PS.55,31 0.1 s, pass energy 30 eV with 90º of electron exit angle. IR-ATR
After 1 hour stirring under argon at RT, dry NP (40 mg) and spectra were recorded on
triethyl amine (TEA, 3.6 x 10-2 mol) were added. The reactants spectrometer in a 4000-400 cm-1 range. The silica nanoparticle
were stirred for 3 hours. Next, grafted NPs were washed with absorption spectra were recorded on UV-Vis-NIR
a
IRAffinity-1S Shimadzu
a
THF until the supernatant was completed colorless and spectrophotometer (Cary 7000) with two lamps (halogen lamp
collected by filtration (Scheme S1, ESI). However, since BDP1 for the Vis-IR region and deuterium lamp for the UV region), a
decomposed under these conditions, an alternative synthesis double monochromator (Littrow) and double diffraction
was performed using as reactant ethyl chloroformate (Acros) grating 1200 lines/mm and an integrating sphere (Internal DRA
and TEA. First the PS (0.027 mmol) was dissolved in dry THF 900).
(25 mL) and ethyl chloroformate (1.6 x 10-4 mol, 15
TEA (1.6 x 10-4 mol, 22
L) were added drop by drop
reaction was carried out under argon at ºC for 30 minutes. tetrabutylammonium fluoride salt (Meth. 1 in Table 1) or 2) by
ꢂ
L) and The total amount of grafted PS was evaluated by two different
The approaches: 1) by dissolving the silica nanoparticles in
ꢂ
.
0
Then, the NPs were added and the reaction mixture was the photometric reading of a previously weighed amount of
stirred at room temperature for 30 minutes. The NPs were grafted nanoparticles in suspension, assuming the same molar
washed with THF until the supernatant was completely clear extinction of the PS in the NP and in solution (Meth. 2 in Table
and collected by filtration.
1). The first method is more accurate, since in the second
approach very stable suspensions are required because the
aggregation and/or flocculation of the nanoparticles will
induce an under-estimation of the absorbance and
consequently of the dye concentration. Accordingly the first
method was applied to NP-RB1 and NP-RB2 since the BDP
chromophores were decomposed in NP-BDP1 and NP-BDP2
after this treatment. Therefore, the BDP loading was estimated
2.4. Grafting of PS on NP surface through OH
groups (NP-RB2 and NP-BDP2)
Grafting of RB2 on surface silanols (NP-RB2). First, RB1 (6 x 10-3
mmol) was dissolved in dry THF (20 mL) and then NHS (0.14
mmol) and EDC (0.1 mmol) was added. After 1 h stirring under
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ARTICLE
by method 2 for these latter samples since they gave relatively improving the stability of the NP suspensions in water for in
stable suspensions.
vitro phototoxicity experiments.
The fluorescence and singlet oxygen measurements were
recorded with an Edinburgh spectrofluorimeter (FLSP920
model) equipped with a 450 W xenon flash lamp. The
fluorescence spectra were corrected for the wavelength
dependence of the detector sensitivity. Radiative decay curves
were recorded with time-correlated single-photon counting
(Edinburgh Instruments, model FL920) using a microchannel
plate detector (Hamamatsu C4878) with picosecond time
resolution. The singlet oxygen quantum yields were
determined by direct measurement of the luminescence at
Acknowledgements
The authors thank the Spanish Ministry of Economy and
Competitiveness, MINECO, (MAT 2014-51937-C3-3, MAT 2014-
51937-C3-2 and MAT2015-68837-REDT) and Basque
Government (IT912-16) for funding this research. VMM
acknowledge Ministerio de Economía
y Competitividad
MINECO for ‘Ramón y Cajal’ contract (RYC-2011-09505). NEE
acknowledges Universidad del País Vasco (UPV-EHU) for a
predoctoral contract and Université de Pau et Pays de l’Adour
(UPPA) together with UPV-EHU for a co-advised doctoral grant.
E. P.-C. wishes to thank CONACyT (Grants 129572 and 123732).
1270 nm with
a
NIR detector integrated in the
spectrofluorimeter (InGaAs detector, Hamamatsu G8605-23)
using as reference RB1 in ACN and the already described 8-
methylthio-2,6-diiodo BODIPY (2I-Thio-BDP) in CHCl3.29
Notes and references
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