Ultrabright BODIPY-Tagged Polystyrene Nanoparticles - American

Article pubs.acs.org/JPCC

Ultrabright BODIPY-Tagged Polystyrene Nanoparticles: Study of Concentration Effect on Photophysical Properties Chloé Grazon,† Jutta Rieger,‡,§ Bernadette Charleux,∥ Gilles Clavier,*,† and Rachel Méallet-Renault*,† †

PPSM, ENS Cachan, CNRS, 61 av Président Wilson, F-94230 Cachan, France Sorbonne Universités, UPMC Univ Paris 06, UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des Polymères, F-75005, Paris, France § CNRS, UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des Polymères, F-75005, Paris, France ∥ Université de Lyon, Université Lyon 1, CPE Lyon, CNRS UMR 5265, C2P2, Team LCPP Bat 308F, 43 Bd du 11 novembre 1918, 69616 Villeurbanne, France ‡

S Supporting Information *

ABSTRACT: Fluorescent nanomaterials are invaluable tools for bioimaging. Polymeric nanoparticles labeled with organic dyes are very promising for this purpose. It is thus very important to fully understand their photophysical properties. New fluorescent core−shell nanoparticles have been prepared. The outer part is a poly(ethylene glycol)-block-poly(acrylic acid) copolymer, and the core is a copolymer of styrene and methacrylic BODIPY fluorophore. The hydrophilic and hydrophobic parts are covalently linked, ensuring both stability and biocompatibility. We prepared nanoparticles with increasing amounts of BODIPY, from 500 to 5000 fluorophores per particles. Increasing the concentration of BODIPY lowers both the fluorescence quantum yield and the lifetime. However, the brightness of the individual particles increases up to 8 × 107. To understand the loss of fluorescence efficiency, fluorescence decays have been recorded and fitted with a mathematical model using a stretched exponential function. This result gives an insight into the fluorophore arrangement within the hydrophobic core.

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developed: physical trapping of dyes in polymeric particles6 and covalent attachment.7 One of the main problems of the first approach is that the fluorophores can leak out of the particles with time. Covalent linking of the fluorophore to the polymer backbone can be achieved either by postmodifying the polymer8 with reactive fluorophores or by copolymerizing fluorescent monomers with a comonomer. As such, rhodamine,9 fluorescein,10 or BODIPY-derived monomers11,12 have been successfully used to prepare fluorescent nano-objects. However, there is one major drawback when organic fluorophores are concentrated in a confined space such as a particle: fluorophore−fluorophore interactions can provoke luminescence quenching.13 This leads to a decrease in the fluorescence lifetime and quantum yield and therefore brightness. The latter is a particularly important parameter in the design of fluorescent nanoparticles because it takes into account both absorption and fluorescence parameters and will determine how the fluorescent nano-objects will be detected in single-particle fluorescence imaging. It is thus important to do extensive photophysical studies of such fluorescent nanoparticles to uncover the origin of the fluorescence decrease and

INTRODUCTION Optical imaging is becoming increasingly attractive in medicine, biology, and biochemistry because it can achieve high spatial and temporal resolution and is noninvasive.1 In addition, fluorescence imaging is a very sensitive technique which can be miniaturized into high sensitivity and cost-effective devices.2 Usually, organic fluorophores are hydrophobic compounds and as such are not soluble in biological media. They can be modified with water-solubilizing groups; however, most of the time this results in a dramatic drop of the fluorescence quantum yield.3 An appealing alternative is to incorporate them in organic or inorganic nanoparticles that can be dispersed in water.1,4 The advantages are numerous because high local concentration of dye can be achieved, leading to lower total amount of material needed for detection of the fluorescence signal. Additionally, multiple fluorophores can be used to achieve bar-coding or multiplex analysis using energy transfer.5 Furthermore, the nanocarrier itself can bring complementary properties such as protection toward photodegradation, water solubility, and, more importantly, biocompatibility while being small enough to penetrate cells. To this end, polymeric nanoparticles have received widespread interest because they benefit from a great variety of synthetic mode, available monomers, and compatibility with organic fluorophores. Two main doping techniques have been © XXXX American Chemical Society

Received: March 20, 2014 Revised: June 3, 2014

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dx.doi.org/10.1021/jp502790w | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

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exclusion chromatography (SEC) using THF as an eluent at a flow rate of 1 mL min−1. For analytical purposes, the acidic functions of the block or statistical copolymers were turned into methyl esters.15 The copolymers were recovered by drying of the aqueous suspensions. After dissolution in a THF/H2O mixture and acidification of the medium with a 1 M HCl solution, they were methylated using an excess of trimethylsilyldiazomethane. Polymers were analyzed at a concentration of 5 mg mL−1 in THF after filtration through 0.45 μm pore size membrane. The SEC apparatus is equipped with a sample delivery module (Viscotek GPCmax) and two columns thermostated at 40 °C (PLgel Mixed; 7.5 × 300 mm2; bead diameter, 5 μm). Detection was made with a differential refractive index detector (Viscotek VE 3580 RI detector) and an ultraviolet−visible (UV−vis) detector (Waters 486 tunable absorbance detector). The Viscotek OmniSEC software (v 4.6.2) was used for data analysis, and the relative Mn and Mw/ Mn were calculated with a calibration curve based on polystyrene standards (from Polymer Laboratories). The z-average particle diameter (named Dz) and the particle size distribution (dispersity factor, named σ), were determined by dynamic light scattering (DLS) of the diluted aqueous dispersions, at an angle of 90° at 20 °C, with a Zetasizer Nano S90 from Malvern, using a 4 mW He−Ne laser at 633 nm. A value of poly below 0.1 is characteristic of a narrow particle size distribution. All calculations were performed using the Nano DTS software. UV−visible spectra were recorded on a Varian Cary (Palo Alto, CA) double-beam spectrometer using a 10 mm path quartz cell from Thuet (Bodelsheim, France). Excitation and emission spectra were measured on a SPEX Fluoromax-3 (Horiba Jobin-Yvon). A right-angle configuration was used. Optical density of the samples was checked to be less than 0.1 to avoid reabsorption artifacts. The relative fluorescence quantum yields ΦF were determined using Rhodamine 590 (ΦF = 0.95 in ethanol) as a reference (error of 15%).16 The fluorescence decay curves were obtained with a time-correlated single-photon-counting method using a titanium−sapphire laser (82 MHz; repetition rate lowered to 4 MHz by a pulsepeaker; 1 ps pulse width; a doubling crystals is used to reach 495 nm excitation) pumped by an argon ion laser from Spectra Physics (Mountain View, CA). The Levenberg−Marquardt algorithm was used for nonlinear least-squares fit with single- or multiexponential functions as implemented in the Globals software (Globals Unlimited, Villa Grove, IL). To estimate the quality of the fit, the weighted residuals were calculated. In the case of single-photon counting, they are defined as the residuals, i.e., the difference between the measured value and the fit, divided by the square root of the fit. χ2 is equal to the variance of the weighted residuals. A fit was said appropriate for χ2 values between 0.8 and 1.2. The average fluorescent lifetime ⟨τ⟩ is calculated as17

potentially propose new architectures or synthetic strategies to circumvent this limitation. Recently, we reported the original synthesis of fluorescent nanoparticles (NP) via a one-pot miniemulsion process, without using surfactants and ultrahydrophobic agents.12 The resulting particles are composed of an amphiphilic triblock copolymer, where poly(ethylene glycol)-block-poly(acrylic acid) constitutes the hydrophilic shell and a copolymer of styrene (S) and BODIPY-derived methacrylate (BDPMA), poly(S-coBDPMA), the core of the particles (Figure 1). One of their

Figure 1. Schematic representation of the fluorescent core−shell nanoparticles.

major features is that the hydrophilic block and the hydrophobic block are covalently linked thanks to the polymerization technique used (reversible addition−fragmentation chain transfer, RAFT), which allows successive growing of the blocks. The particles have a hydrodynamic diameter of 70 nm, are highly stable in water, and include approximately 5000 fluorophores each. The fluorescence lifetime and the quantum yield of these NP aqueous dispersions were lower than those of the corresponding monomeric fluorophore dissolved in toluene. It was determined from the polymerization kinetics data that BDPMA was consumed quicker than styrene, leading to a gradient of composition along the resulting polymer chain: the content of fluorophore is higher in the first portion of the hydrophobic block, i.e., the part next to the hydrophilic block constituting the particle shell, and should therefore be concentrated at the periphery of the nanoparticle core. Thus, we tentatively attributed the observed weaker fluorescence of the BODIPY in the NP to fluorophore−fluorophore interactions. To gain deeper insight into the origin of the loss of fluorescence efficiency, four different samples of nanoparticles have been prepared with an overall structure similar to the reported one but with different quantities of BDPMA in their core. We have compared the spectroscopic properties of the BODIPY monomer in solution, the nanoparticles aqueous dispersions, and solutions of the individual polymer chains which constitute them. A model which uses stretched exponentials14 was developed to fit the fluorescence decays. Thanks to this approach, it was possible to link the fluorescence properties of the nanoparticles and the spatial distribution of the fluorophores within the hydrophobic core.

∞

⟨τ ⟩ =

∫0 tI(t ) dt ∞

∫0 I(t ) dt

(1)

where t is the time and I(t) is the intensity of fluorescence decay. The radiative decay rate kr is calculated as

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MATERIALS AND METHODS Instrumentation. The number-average molar mass Mn, weight-average molar mass Mw, and the molar mass distribution (polydispersity index Mw/Mn) were determined by size

kr = B

ϕF ⟨τ ⟩

(2) dx.doi.org/10.1021/jp502790w | J. Phys. Chem. C XXXX, XXX, XXX−XXX


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Table 1. Main Fluorescent Properties of the FNPs with Variable Amount of BDPMA Copolymerized in Their Core water

toluene

exp

[BDPMA]a (mol/LS)

nBDPMAb

ΦF

⟨τ⟩c (ns)

kr (× 10−7 s−1)

ΦF

⟨τ⟩c (ns)

kr (× 10−7 s−1)

BDPMA FNP1 FNP2 FNP3 FNP4

− 0.02 0.08 0.17 0.23

− 0.3 1.1 2.1 3.0

− 0.56 0.39 0.24 0.20

− 5.8 4.3 3.2 2.8

− 9.7 10 7.5 7.1

0.69 0.63 0.68 0.69 0.47

4.9 4.9 4.9 4.9 4.8

14 13 14 14 9.8

BDPMA concentration in the fluorescent core of the FNPs in moles per liter of styrene. bAverage number of BDPMA per polymer chain (PEO-bPAA-b-P(S-co-BDPMA)). nBDPMA = [BDPMA]i/[RAFT]i, where [BDPMA]i and [RAFT]i are the concentration of BDPMA monomer and RAFT agent at the initial state of the polymerization, respectively, knowing that the conversion of BDPMA is close to 100% (see Table S1 in Supporting Information). cAverage fluorescent lifetime estimated with eq 1 (λexc = 495 nm, λF = 543 nm for the FNPs in water and polymer chains in toluene and λF = 540 nm for BDPMA). a

where ΦF is the fluorescence quantum yield and ⟨τ⟩ is the average fluorescence lifetime. Materials. BODIPY monomer with a phenyl methacrylate function (BDPMA) and PEO-b-PAA-TTC-C12 macro-RAFT agent were obtained as described elsewhere.12 Styrene (≥99%, Sigma-Aldrich) was purified by distillation under reduced pressure. Solvents (Carlo Erba) were of synthetic grade for synthesis and spectroscopy grade for physicochemical analysis. Synthesis of Fluorescent Nanoparticles. RAFT polymerizations of styrene and BDPMA (0.15 to 2 mol %) were performed in the presence of PEO45-b-PAA10-TTC-C12 (Mn = 3.1 kg/mol; Mn,SEC = 4.1; Mw/Mn = 1.1) macroRAFT agent in a one-pot phase inversion miniemulsion process as described elsewhere.12

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Figure 2. Absorption (full lines) and fluorescent emission (dotted lines) spectra of BDPMA recorded in toluene (gray) and of the dispersion of FNP4 in water (black).

RESULTS AND DISCUSSION than that in the monomer spectra. This effect has already been reported for BODIPY confined in a rigid polymer matrix.19 Indeed, the more rigid a molecule is the more pronounced will be its vibrational signature in a UV−vis spectrum. In this case, BDPMA entrapped in polystyrene has a more constrained geometry leading to an increased vibrational shoulder. The emission spectra of the BODIPY nanoparticle dispersions (S F = 543 nm) are also highly similar to the spectrum of the free monomer in toluene (S F = 540 nm). A slight red shift is observed from the free monomer to the monomer copolymerized with styrene and assembled in the NP core. The same observation has already been made for BODIPY confined in a rigid polymer matrix.19,20 The fluorescence decays of the monomer in toluene and the FNP in water were recorded at 543 nm (S ex = 495 nm, Figure S2 in Supporting Information). The monomer’s decay could be fitted by a single-exponential function in contrast to the FNP decay. The fluorescence quantum yields ϕF and intensity-averaged fluorescence lifetimes ⟨τ⟩ (eq 1) of the fluorescent NPs were determined and compared as a function of the average number of BODIPY molecules per polymer chain. As can be seen in Figure 3, the quantum yields and the average fluorescence lifetimes decrease from 0.56 to 0.20 and 5.8 to 2.8 ns, respectively, when the concentration of BDPMA increases in the core of the fluorescent NPs from 0.3 to 3 BDPMA per chain on average. To understand this phenomenon, the radiative decay rate kr have been estimated (eq 2 and Table 1). When the average number of BDPMA per chain varies from 0.3 to 3, the kr is divided only by 1.4 (from 9.7 × 107 to 7.1 × 107 s−1). Therefore, with increasing amount of BDPMA in the FNPs, the kr remains almost constant whereas the fluorescence quantum yield decreases nearly three times, meaning that a

Recently, our team succeeded in synthesizing core−shell nanoparticles via RAFT- controlled radical polymerization. The obtained particles possess a hydrophilic shell made of a double hydrophilic block copolymer of poly(ethylene glycol) and poly(acrylic acid) covalently linked to a hydrophobic core of polystyrene chains to which a fluorescent monomer is incorporated by copolymerization (Figure 1). BODIPY has been chosen as a hydrophobic fluorophore as it exhibits attractive spectroscopic characteristics such as an emission spectrum tunable from green to red and high fluorescence quantum yields.18 The monomer is a BODIPY derivative with a phenyl methacrylate polymerizable function, named BDPMA (Figure S1 in Supporting Information). Four different particles have been synthesized with the same architecture but different BDPMA content. In all cases, the conversion for both styrene and BDPMA was close to 100% (Table S1 in Supporting Information). All nanoparticles had a size around 70 nm and, considering a total number of 120 monomer units in the hydrophobic block, the average number of BDPMA per chain varies from 0.3 to 3. These numbers were calculated considering that the initial BDPMA concentration in styrene in the polymerization ranged from 0.02 mol/LS to 0.23 mol/LS (from 1 to 10 wt % related to styrene) and that the monomer conversions were 100% (Table 1). Absorption and fluorescence spectra of BDPMA recorded in toluene and of FNP4 recorded in water are shown in Figure 2. The spectra of dispersions FNP1 to FNP4 (FNP1−4) are identical in shape and position. Absorption spectra of the aqueous dispersion of the fluorescent nanoparticles have the same maximum as that of the BDPMA monomer in toluene (S abs = 528 nm). The vibrational shoulder of the S0 → S1 band (around 500 nm) in the particle spectra is more pronounced C



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was estimated with the Strickler−Berg equation22 and found to be equal to 6.3 ns. The intercept value can then be interpreted as the lifetime of an isolated BDPMA in a rigid polymer matrix. It has been observed previously that BODIPY dispersed in or attached to a rigid matrix (such as polymers) below their glass transition temperature have a longer lifetime than that in solution because of lower probability of certain nonradiative deactivation pathways, such as solvent transfer.19 To gain deeper understanding into the origin of the loss of fluorescence for the NPs possessing higher quantities of BODIPY, individual polymer chains which constitute the FNPs were studied after dispersion in toluene. In practice, NPs were precipitated in water acidified with HCl 1 M (pH ∼1.5) and then extracted with toluene. The phase transfer could be visually monitored because the initial pink water phase became colorless, whereas the organic phase became pink. Then, absorption and fluorescence spectra of the polymer chains were recorded in diluted solution in toluene (Table 1). For the polymer chains constituting FNP1−3, the fluorescence quantum yield kr in toluene is identical to that of the monomer in toluene. Furthermore, their fluorescence decays (Figure S3 in Supporting Information) could be fitted with a single exponential with the same lifetime as BDPMA. Those results indicate that the BDPMA units in the polymer chains behave spectroscopically like the diluted monomer when the polymers are not sequestrated in a nanoparticle. On the other hand, for FNP4 where the BDPMA monomer is the most abundant, the fluorescence quantum yield is lower than the BDPMA in toluene by a factor of 1.5. The fluorescence decay cannot be fitted with a single exponential anymore, and the kr of those polymer chains is lower than that for the free monomer or other polymer chains with smaller amounts of BDPMA. However, the intensity averaged lifetime remains close to the lifetime of the monomer because the short component contribution to the decay is small (less than 20%). Hence, the main origin of the fluorescence decrease does not come from intrachain interactions between fluorophores because it starts to be detected only in the most concentrated ones (FNP4). A more detailed analysis of the fluorescence decays of the FNP was thus conducted. Time-resolved fluorescence is commonly used to acquire knowledge of the interactions within an assembly of fluorophores. For pure and isolated fluorophores, the fluorescence decay can be adjusted by a single monoexponential function. For assemblies of fluorophores of the same or different nature, data are usually fitted with a sum of discrete exponentials. Nevertheless, for fluorophores confined in DNA,23 polymer24 or sol−gel matrixes25 for example, the use of a continuous distribution of decay times can be more appropriate.26 The fluorescence decays of FNP1−4 have been analyzed and adjusted with different methods. The decays are complex and can only be fitted satisfactorily with a sum of three exponentials. However, the physical meaning of this sum of three exponentials is not obvious because only one fluorophore is present. A more realistic model for fluorophores immobilized in a matrix is the use of the stretched exponentials, also called Kohlrausch functions.14 Kohlrausch functions can be expressed as

Figure 3. Evolution of the fluorescence quantum yield ϕF (squares) and of the average lifetime ⟨τ⟩ (triangles; eq 1) of the different FNP recorded in water as a function of the average number of BDPMA per polymer chain. The values for 0 BDPMA per chain correspond to the fluorescence quantum yield and lifetime of the BDPMA monomer in toluene.

concentration-quenching phenomenon becomes more and more important and is finally predominant. The inverse of the average lifetime was plotted as a function of the average number of BDPMA per polymer chain (Figure 4). A straight line is obtained (R2 = 0.99) with a slope of 0.08

Figure 4. Evolution of the inverse of the average lifetime ⟨τ⟩−1 of the four FNPs (FNP1−4) in water with the average number of BDPMA per polymer chain.

and a y-intercept of 0.16 ns−1. This behavior and the almost constant value for kr in the series suggest that the loss of fluorescence comes from concentration. Nevertheless, the presence of nonluminescent complexes (due to concentration increase) is not clearly seen because the position and shape of both absorption and emission spectra are constant for all particles in the FNP1−4 series and are similar to the monomer spectra. We have previously shown that BDPMA is consumed faster than styrene in the course of their copolymerization.21 Hence, as explained in the introduction, the BODIPY fluorophores are predominantly incorporated early in the growing polymer chains; therefore, when their concentration in the NP is increased, some fluorophores could be close enough in the outer part of the NP to form nonfluorescent aggregates which act as quenchers or traps of the fluorescence. The linear regression of the plot in Figure 4 intercepts the origin at 0.16 ns−1. This value corresponds to a lifetime of 6.3 ns which is longer than that of BDPMA in diluted solution in toluene (4.9 ns). The natural radiative lifetime τ0 of BDPMA

⎛ t ⎞β I(t ) = I0 exp⎜ − ⎟ = ⎝ τ0 ⎠ D

∫0

∞

f (k) exp( −kt ) dt (3)



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Figure 5. Fluorescence decays of the four FNP (FNP1−4) with different amounts of BDPMA. λexc = 495 nm; λF = 543 nm. Experimental data (points) and fits (solid lines) with eq 4.

noteworthy that the value of τ2 is very close to the one extrapolated at infinite dilution from the plot in Figure 4 (6.3 ns). Hence the assumption that a population of dispersed fluorophores exists within the FNPs, which behaves independently, is very likely. The β exponent is consistent with results found by Winnik et al. for fluorescent monomer in PS matrix (0.9).27 As can be seen in Table 2, when the average number of

where 0 < β ≤ 1 and τ0 and 1/k are lifetimes. Thus, when β = 1, the decay follows a single-exponential law. Stretched exponentials have been used to describe the fluorescence decays of donors in system where resonant energy transfer takes place. In those cases it was found that β adopts special values.14 However, in our case, it has not been possible to fit the experimental decays with a pure Kohlrausch function (eq 3) because all of those mathematical expressions decay too fast compared to our decay profiles. On the basis of the structure of the FNPs, a model was developed. It consists of a sum of a stretched exponential (Kohlrausch) and a discrete exponential: ⎛ t ⎞ ⎛ t ⎞β I(t ) = a1 exp⎜ − ⎟ + a 2 exp⎜ − ⎟ ⎝ τ2 ⎠ ⎝ τ1 ⎠

Table 2. Fluorescent Decay Fitting Parameters (eq 4) of the FNPs with Different Amounts of BDPMA, Recorded in Water (λexc = 495 nm, λF = 543 nm), with β and τ2 Optimized for the Four Decays and τ1, a1, and a2 variables. β = 0.88, τ2 = 6.0 ns, and overall χ2 = 1.21

(4)

The stretched exponential should model the emission of the fluorophores which are located close to the interface between the hydrophobic core and the hydrophilic shell. Those molecules are presumably interacting with each other and subject to concentration quenching. Therefore, their fluorescence decay should be fast and complex. The discrete exponential will take into account the long tail of the decay coming from individual fluorophores probably located deeper in the center of the FNP. Those molecules are then far enough apart from each other to behave independently. With this expression, we have been able to fit the decays of FNP1−4 (Figure 5). The τ2 and β parameters were kept constant for all decays, while the others were adjusted. Indeed, it was assumed that isolated fluorophores should all have the same fluorescent behavior (τ2 constant) and that the heterogeneity of the interacting fluorophores should be similar from one sample to another (β constant). The values found for these two parameters were τ2 = 6.0 ns and β = 0.88. It is

exp

[BDPMA]a (mol/LS)

FNP1 FNP2 FNP3 FNP4

0.02 0.08 0.17 0.23

nBDPMAb τ1 (ns) 0.3 1.1 2.1 3.0

5.4 2.9 1.8 1.6

a1

a2

χ2

0.21 0.95 0.93 0.94

0.79 0.05 0.07 0.06

1.09 1.46 1.08 1.22

a BDPMA concentration in the fluorescent core of the FNPs in moles per liter of styrene. bAverage number of BDPMA per polymer chain (PEO-b-PAA-b-P(S-co-BDPMA)). nBDPMA = [BDPMA]i/[RAFT]i, where [BDPMA]i and [RAFT]i are the concentration of BDPMA monomer and RAFT agent at the initial state of the polymerization, respectively, knowing that the conversion of BDPMA is close to 100% (see Table S1 in Supporting Information).

BDPMA per polymer chain varies from 0.3 to 3, the lifetime τ1 decreases from 5.4 to 1.6 ns. Moreover, the pre-exponential factor a1 is equal to 0.21 for the FNP1 (the one with the lowest amount of BDPMA) and is equal to 0.93−0.95 for the three other fluorescent NPs (FNP2−4). The pre-exponential factor a2 is equal to 0.79 for the FNP1 and to 0.05−0.07 for FNP2−4. Therefore, the FNP1 decay follows almost a pure monoE



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B = NBDPMAεBDPMA ΦF

exponential decay, whereas FNP2−4 follow almost pure stretched exponential decays. Those results point to the presence of two different populations of fluorophores within the nanoparticles: (i) isolated BDPMA, probably located in the deeper part of the particles, with a lifetime τ2 = 6 ns; and (ii) BDPMA concentrated at the hydrophobic−hydrophilic interface and located on different polymer chains possessing a distribution of lifetimes, which become shorter when their concentration increases. Nonluminescent interchain complexes may form within this later population of BODIPY. However, it was not possible to detect their presence in the absorption or fluorescence emission spectra, and their concentration should be low. On the other hand, the fluorophores located at the periphery of the FNP are close enough to establish efficient energy transfers among them. 28 To characterize this energy transfer between fluorescent BDPMA, time-resolved anisotropy decays were recorded. Unfortunately, transfer rates were faster than the time resolution of our set up. The transfer rate is thus larger than ≈3 × 1010 s−1, corresponding to over 200 energy transfers during the excitation time. Thus, even if there is a small number of nonfluorescent complexes in the FNPs they can act as efficient fluorescence traps because of rapid energy transfer. The distribution function f(k) of a stretched exponential is the inverse Laplace transformation of I(t) and has only an analytical result for β = 0.5. When β ≠ 0.5, different models have been proposed to obtain the distribution function f(k). An estimation of f(k) has been proposed by Berberan-Santos et al. When β > 0.5, the distribution function can be expressed as14 f (k) = τ0

B β 1 − 2 /1 − β

(kτ0)

(6)

where εBDPMA is the molar coefficient extinction of BDPMA at 528 nm, NBDPMA the number of BDPMA per particle (Table S1 in Supporting Information), and ΦF the quantum yield of the fluorescent NPs. εBDPMA was taken to be equal to the one determined for BDPMA in toluene (73 000 mol−1 cm−1 L) considering that this solvent should have properties similar to polystyrene. The NBDPMA can be estimated as the number of BDPMA per polymer chain nBDPMA multiplied by the aggregation number of the NPs. The aggregation number Nagg is the number of polymer chains per nanoparticle and was calculated as follows: mρ n V Nagg = chain = S nchain = 4 S3 nchain nFNP VFNP πr (7) 3 where nFNP is the total number of fluorescent nanoparticles in the synthesis batch, nchain the total number of growing chains (which is equal to the number of macroRAFT agents assuming they are all incorporated in the NPs), VFNP the volume of one fluorescent nanoparticle calculated form DTEM determined by transmission electron microscopy (TEM), VS the total volume of styrene in the synthesis, r the core radius of the nanoparticles determined by TEM, ρS the polystyrene density, and m the total mass of styrene in the synthesis. For all nanoparticles, Nagg is close to 1750 (±250). As can be seen in Figure 7, even if the fluorescence quantum yield decreases when the amount of BDPMA increases in the

⎡ (1 − β)β β /1 − β ⎤ ⎥(1 + C(kτ0)δ ) exp⎢ − ⎢⎣ (kτ0)β /1 − β ⎥⎦ (5)

where B and C are parameters depending on the value of β and δ = [β(β − 0.5)]/(1 − β). The β exponent is proportional to the inverse of full width at half-maximum of the lifetime distribution. The lifetimes distribution function f(k) is plotted in Figure 6. It is clear that when the concentration of BDPMA

Figure 7. Evolution of the brightness B (circles) and of the fluorescence quantum yield ϕF (squares) of the different FNP in water as a function of the average BDPMA per polymer chain. The open circle (○) corresponds to the fluorescence quantum yield of the monomer BDPMA in toluene.

core of the fluorescent NPs (from 525 to 5250), the brightness increases by a factor of 4. For the FNP4 containing 5250 BDPMA, the brightness is 1000 times higher than a single BODIPY dye. Moreover, it is 200 to 2000 times higher than that of classical quantum dots29 or similar fluorescent NPs.8b

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Figure 6. Distribution of lifetimes k−1 determined with eq 5 for FNP1−4. The Dirac number corresponds to 1/τ2 (τ2 = 6.0 ns).

CONCLUSION Self-stabilized polymer nanoparticles containing different concentrations of a phenyl methacrylate BODIPY monomer were designed and characterized. The absorption and emission spectra of the fluorescent nanoparticles in water are very similar to those of the BDPMA monomer in toluene. Experimental fluorescence decay profiles of FNPs are shorter and more complex than that of BDPMA in toluene solution, which follows a single exponential. Both fluorescence quantum yield

increases in the hydrophobic core of the NP, the distribution of lifetimes becomes larger, reflecting an increasing inhomogeneity of interactions within that population. Brightness. Finally, the average brightness of individual FNPs has been estimated because it is one of the most important parameters for biological imaging. The brightness B was calculated as F



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*E-mail: [email protected].

and intensity-averaged fluorescence lifetime decrease when the BDPMA concentration increases (from 0.3 to 3 fluorophores per polymer chain). Looking at the deactivation rate constants, it appeared that nonradiative deactivation pathways become more and more predominant when the BDPMA concentration increases, whereas the radiative deactivation remains almost constant. This was attributed to an increasing probability of fluorophore−fluorophore interactions with increasing BDPMA concentration in the FNP because of their spatial rapprochement in the individual polymer chains and/or in the particles. Interestingly, individual polymer chains constituting the FNPs show fluorescence efficiency after dispersion in toluene that is the same as that of the monomer except for the most concentrated one (nBDPMA per chain = 3). This means that, when gathered in FNPs, intrachains interactions, i.e., interactions of BOPIPY molecules on the same polymer chain, are a minor phenomenon and cannot account for the loss of fluorescence efficiency. A model was developed to fit the experimental FNP decays using a sum of a monoexponential function and a stretched exponential one. Indeed, we assumed the existence of two populations: isolated fluorophores with a constant lifetime (monoexponential function) and interacting fluorophores concentrated at the hydrophilic−hydrophobic interface with a distribution of lifetimes (stretched exponential). The isolated fluorophore lifetime was found to be close to the extrapolated value at infinite dilution in a polymer matrix. The lifetimes of interacting BDPMA diminishes with fluorophore concentration. These results are in full agreement with what is known from polymerization kinetics data and the relative reactivity of BDPMA and styrene. It was reported that because of differences in reactivity during the copolymerization with styrene, a large fraction of BDPMA molecules is mainly concentrated at the hydrophilic−hydrophobic interface and a smaller one is located in the deeper part of the NP core.21 In a related study, we have shown that the nature of the polymerizable groups on the BODIPY framework is crucial for the fluorophore distribution along the polymer chain and thus its distribution in the FNP, which has an impact on the fluorescence quantum yield.21 These results highlight a fundamental role of the fluorophores’ spatial location in the FPNs on the photophysical properties. Most importantly and despite the decrease of the fluorescence properties with increasing fluorophore concentration, it must be noted that the brightness of the FNPs is concomitantly strongly increasing, reaching values hundreds or thousands of times higher than standard quantum dots or previously reported FNPs. This makes the nanoparticles very valuable objects for bioimaging considering that they are heavy-metal free, do not contain any surfactant, and can be further postfunctionalized thanks to the poly(acrylic acid) block. Current work aims at modifying the fluorophore in order to change the color of the FNPs and to use them for imaging of eukaryotic cells and bacteria.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Gaëlle Pembouong (LCP) for technical support on SEC analyses, Arnaud Brosseau (PPSM) for technical support on fluorescence spectroscopy measurements, and Dr. Aniss Bendjoudi for help on mathematical analysis. Dr. Thomas Gustavsson is greatly acknowledged for helpful discussions. B.C. acknowledges the Institut Universitaire de France for her nomination as a senior member.

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ABBREVIATIONS BDPMA 2,6-diethyl-4,4-difluoro-8-(4-(methacryloyloxy)phenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene; RAFT reversible addition−fragmentation chain transfer; PS polystyrene; PEO poly(ethylene oxide); PAA poly(acrylic acid) ; P(S-co-BDPMA) poly(styrene) copolymerized with BDPMA monomer; FNP fluorescent nanoparticles

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ASSOCIATED CONTENT

S Supporting Information *

Molecular structure of BDPMA, synthetic details and characteristics of the nanoparticles, and fluorescence decays of monomer and nanoparticles in water and toluene. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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