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NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging
Dyes and Pigments xxx (2014) 1e8
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging Tânia Ribeiro, Sebastian Raja, Ana S. Rodrigues, Fábio Fernandes, Carlos Baleizão*, José Paulo S. Farinha* CQFM e Centro de Química-Física Molecular and IN e Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2014 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online xxx
We describe the preparation of very bright and photostable fluorescent silica nanoparticles (SiNPs), with emission in the visible or in the near infrared (NIR). The nanoparticles can be prepared with diameters of 30e300 nm with low size dispersity, and incorporate perylenediimide (PDI) dyes modified with alkoxysilane groups that react with the silica structure. One of the dyes (PDIvis) emits in the green and the other (PDInir) in the NIR (with fluorescence quantum yields of 90% and 24%, respectively). Silica nanoparticles containing these dyes were efficiently internalized in HEK293 cells with low toxicity and reveled very high photostability, showing good potential for use as a platform for in vivo laser scanning bioimaging applications. The NIR emitting nanoparticles, in particular, can be used in multi-color imaging, even in cells expressing high levels of fluorescent proteins and/or co-stained with different fluorescent dyes (most of which commonly emit at wavelengths lower than the NIR). Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Fluorescent silica nanoparticles Perylenediimide NIR dyes Laser scanning confocal fluorescence microscopy Bioimaging RGD
1. Introduction Nanoparticle-based fluorescent imaging agents offer many advantages over molecular dyes, such as improved photo-robustness of the encapsulated dyes, the ability to carry large payloads of the optical probes, and the possibility of decorating the surface with specific groups. Additionally, nanoparticles can also carry and release drugs, leading to the development of different systems for use in combined diagnostic and therapeutic (theranostic) applications [1]. Silica nanoparticles (SiNPs), in particular, have been successfully used in nanomedicine, [2,3] as supports or carriers in drug delivery [4] and imaging [5]. The increasing interest in SiNPs is due to the possibility of tuning their diameter and porosity, functionalize the surface, load large amounts of fluorophores, drugs or other molecules, and control if and how the cargo is released [6]. Perylenediimide based dyes (PDIs) have many applications [7e 9] due to their typically high photochemical stability and extinction coefficients, and large fluorescence quantum yields [10]. The
* Corresponding authors. Tel.: þ351 218419221. E-mail addresses: [email protected] (C. Baleizão), farinha@ tecnico.ulisboa.pt (J.P.S. Farinha).
photophysical properties of PDIs can be modulated by introducing appropriate substituents in the imide group (to change solubility or allow immobilization) or in the perylene core (affecting the electronic and optical properties) [11]. By incorporating PDI dyes in SiNPs, one can obtain higher brightness, better photostability (due to lower oxygen concentration and diffusivity inside the nanoparticles) and also widen the range of solvents/materials in which they can be used by straightforward surface modification of the nanoparticles. We have synthesized two different PDI derivatives modified with alkoxysilane groups for incorporation into silica nanostructures (Scheme 1), one with emission in the green region of the spectrum (530e600 nm) and excitation at 450e530 nm (PDIvis), and the other with excitation in the red and emitting in the near infrared (NIR) region (PDInir) [12,13]. The dyes were incorporated in SiNPs (Scheme 2) with diameters at the limit of the optical resolution (ca. 300 nm, l-SiNP) and also in smaller nanoparticles (ca. 30 nm, s-SiNP), for testing in laser scanning confocal fluorescence microscopy (LSCFM) bioimaging. Due to the increased brightness and photostability of the nanoparticles relative to the isolated dyes, their biocompatibility (unaffected cell viability) and efficient cell internalization, these nanoparticles are very promising for in vivo laser-scanning imaging applications.
http://dx.doi.org/10.1016/j.dyepig.2014.03.026 0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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2. Materials and methods 2.1. Materials
Scheme 1. Structure of the perylenediimide (PDI) derivatives, one with emission in the visible region of the spectrum (PDIvis) and the other emitting in the NIR (PDInir).
Absolute ethanol (Panreac, 99.5%), ammonium hydroxide solution 25% (Fluka), tetraethyl orthosilicate (TEOS, Aldrich, 98%), N-(3dimethylaminopropyl)-N0 -ethylcarbodiimide (EDC, Aldrich, 97%), (3-aminopropyl)trietoxysilane (APTES, Aldrich, 98%) and a RGD oligopeptide (Gly-Arg-Gly-Asp-Ser-Pro, Aldrich) were used as received. Commercial toluene and dimethyl sulfoxide (DMSO) were distilled over calcium hydride before use. Deionized water from a Millipore system Milli-Q 18 MU cm was used for fluorescence measurements and endocytosis experiments. Perylenediimide derivatives, PDIvis and PDInir were synthesized according to the literature [11,12]. Silica nanoparticles doped with both PDI derivatives were synthesized by the Stöber method [26]. Wheat germ agglutinin (WGA) coupled to Alexa Fluor (AF)-594, Hoechst 33258 and DiIC18(5) were purchased from Invitrogen (Carlsbad, CA). 2.2. Synthesis of the fluorescent silica nanoparticles
Furthermore, the possibility of using near infrared (NIR) light (750e1400 nm) instead of lower wavelength (visible) light presents several advantages for optical bioimaging applications [14]. In the NIR region there is less light scattering than in the visible, light absorption and autofluorescence by biological tissues is lower [15] and therefore NIR light has higher tissue penetration [16]. Also, NIR dyes can be excited using less expensive lasers (He:Ne or red diode lasers) and can be used in systems labeled with multiple dyes, since most of the dyes presently used for fluorescence imaging (including most fluorescent proteins) emit at lower wavelength, in the visible region [17]. In spite of the advantages of using NIR dyes for fluorescence bio-imaging and of encapsulating fluorescence dyes in silica nanoparticles, only a few examples of functionalized NIR dyes for incorporation into silica structures, mostly based in carbocyanides, have been described [18e20]. The diameter of the nanoparticles can easily be tuned during synthesis and their surface can be modified by standard procedures for better biocompatibility and/or to achieve targeting to specific types of cells or organelles inside the cell [21,22]. Those strategies include the incorporation of polymer chains (either through surface initiated polymerization or by covalent immobilization of chains), charged species (via covalent attachment), biomacromolecules (as peptides, DNA, antigens), etc. Here, we tested the surface modification of the nanoparticles with an arginineeglycineeaspartic acid (RGD) oligopeptide, an integrin-binding motif known to promote cellular adhesion and proliferation [23]. Since some integrins are specifically expressed in tumor endothelia and certain tumor cells, RGDs can provide tumor-homing properties [24]. Therefore, by decorating the SiNPs surface with a RGD motif, the particles are expected to show enhanced specificity in delivering anticancer drugs and contrast agents for cancer diagnosis and therapy [25].
Water, absolute ethanol, and ammonia solution were mixed in a plastic flask, and the mixture stirred at 30 C. TEOS and PDI were dissolved in absolute ethanol and added at the same time to this mixture. The reaction was kept for 24 h at 30 C with magnetic stirring, after which the nanoparticles were centrifuged at 10,000 rpm for 3 cycles of 30 min, and either dispersed in ethanol or dried in vacuum. The volumes (in mL) of ammonia:TEOS:ethanol:water used were: 1.5:4.5:110:9 and 30 mg of PDIvis for the smaller visible light-emitting nanoparticles s-SiNP-PDIvis; 7.6:3.1:100:13 and 20 mg of PDIvis for large visible light-emitting nanoparticles l-SiNP-PDIvis; and 2.5:1:33:6 and 2.2 mg of PDInir for the NIR-emitting nanoparticles SiNP-PDInir. 2.3. Surface modification of the smaller silica nanoparticles with a RGD oligopeptide Fluorescent and non-fluorescent silica nanoparticles (0.040 g sSiNP-PDIvis or s-SiNP, the later obtained with the same procedure but without using PDIvis) were surface functionalized with APTES (93 mL, 0.4 mmol) in toluene (2 mL) and the reaction was kept under reflux with a magnetic stirring for 24 h at 125 C. The nanoparticles were then recovered by centrifugation at 15,000 rpm (20 min), washed three times with ethanol and dried over night at 40 C. The resulting nanoparticles, s-SiNP-PDIvis-APTES and s-SiNP-APTES (0.033 g) were then mixed with RGD oligopeptide (0.0033 mmol), EDC (2 mL, 0.012 mmol) in distilled DMSO (1 mL) and the mixture stirred at 25 C for 24 h. The mixture was centrifuged at 15,000 rpm (20 min), washed three times with ethanol and the recovered products, s-SiNP-PDIvis-RGD and s-SiNP-RGD were dried over night at 40 C.
Scheme 2. Incorporation of PDI derivatives in silica nanoparticles (SiNPs) by covalent attachment to the silica network during synthesis by a modified Stöber method.
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
T. Ribeiro et al. / Dyes and Pigments xxx (2014) 1e8
3
D
B
Relative frequency
Relative frequency
A 0,4
0,2
0
Relative frequency
26
32 38 44 Diameter (nm)
E
0.6 0.4 0.2
0
C
20
250
300 350 400 Diameter (nm)
F
0.6 0.4 0.2 0
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280 310 Diameter (nm)
Fig. 1. TEM images of s-SiNP-PDIvis (A), l-SiNP-PDIvis (B) and SiNP-PDInir (C), with corresponding diameter distributions (D, E and F, respectively). Scale bars 200 nm.
2.4. In vivo tests HEK293 cells were cultured in an atmosphere of 5% CO2/95% air at 37 C. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY) was used. Transfection was performed in serum-free medium with pEGFP-N1 (Clontech, Palo Alto, CA), and 24 h after cells were seeded in 8 well ibidi m-slides (Martinsried, Germany). Gencarrier-2 from Epoch Biolabs (Sugar Land, TX, USA) was used as the transfection reagent. Transfection medium was removed after 6 h and
Table 1 Photophysical parameters of the dyes PDIvis and PDInir and the fluorescent nanoparticles SiNP-PDIvis and SiNP-PDInir in 1,4-dioxane.
PDIvis SiNP-PDIvisc PDInir SiNP-PDInir a b c
labs max
lemi max
(nm)
(nm)
521 e 685 e
530 528 741 734
3
(M1cm1)a
56,162 e 41,700 e
FF
Brightnessb (104 M1cm1)
s (ns)
0.90 e 0.24 e
5.05 e 1.00 e
4.3 4.4 4.0 3.8
At the absorption maxima. The product between the absorptivity and the quantum yield. Values obtained for s-SiNP-PDIvis and l-SiNP-PDIvis.
cells were incubated with nanoparticles (250 mg/mL) in complete medium for another 12 h. Immediately before imaging, cells were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (AF594-WGA) (5 mg/mL in PBS), Hoechst 33258 (2 mg/mL in PBS) or DiIC18(5) (5 mM in PBS) at 37 C for 10e20 min. Excess dye was removed by washing 3 times with culture medium. 2.5. Cell viability tests Cell viability in the presence of different concentrations of nanoparticles was assessed through the lactate dehydrogenase (LDH) activity in extracellular medium. LDH activity was measured with the Citotoxicity Detection Kit Plus from Roche (Mannhein, Germany) on a BMG POLARstar Optima plate reader (BMG Labtechnologies, Offenburg, Germany). 2.6. Methods Laser scanning confocal fluorescence microscopy (LSCFM) images were obtained on a Leica TCS SP5 laser scanning microscope (Leica Mycrosystems CMS GmbH, Mannheim, Germany) using an inverted microscope (DMI6000) and a HCX PL APO CS 1.20 W 63.3 waterimmersion apochromatic objective (63.3 magnification and 1.2
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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numerical aperture). Imaging and image spectral analysis used either the 488 nm line of an argon ion laser (for PDIvis, 21.4 mW at the sample surface) or the 633 nm line of a He:Ne laser (for PDInir, 325 mW at the sample surface). The laser light intensity was controlled by an acoustic-optical filter system. The laser power at the sample was measured with a PM100D optical power meter with an S120C sensor (Thorlabs, NJ). The fluorescence emission was collected at selected wavelengths, using the tunable system and beam splitter of the Leica TCS SPC5. The laser power and photomultiplier tube gain were constant for all measurements. The offset was chosen such that the photon counts outside the sample were negligible. The pinhole was always set at 1 Airy unit to discriminate stray light from out-of-focus plans. Images obtained at a resolution of 512 512 pixels were processed with the software ImageJ (http://rsbweb.nih.gov/ij/). Emission spectra were obtained in 500 nm regions of the images, with 10 nm steps and a bandwidth of 10 nm. The data was processed using the Leica Application SuiteAdvanced Fluorescence software to obtain the spectra in different regions with a 500 nm diameter in each image. TEM images were obtained on a Hitachi transmission electron microscope (Model H-8100 with a LaB6 filament) with an accelerator voltage of 200 kV. One drop of the nanoparticles dispersion in ethanol was placed on a carbon grid and dried in air before observation. The absorption spectra were recorded on a Shimadzu UV3101PC UV-vis-NIR spectrophotometer, and the fluorescence measurements were obtained on a Horiba Jobin Yvon Fluorolog 322 spectrofluorometer. For the quantum yield, Rhodamine B in ethanol was used as standard [27]. The fluorescence emission spectra were corrected to the photomultiplier response. Timeresolved picosecond fluorescence intensity decays were obtained by the single-photon timing method in a setup consisting of a diode-pumped solid state Nd:YVO4 laser (Milennia Xs, Spectra Physics) synchronously pumping a mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, with tuning range 700e1000 nm, output pulses of 100 fs, and 80 MHz repetition rate that can be reduced to 4 MHz by a pulse picker) or a cavity dumped dye laser (701-2, Coherent, delivering 3e4 ps pulses of ca. 40 nJ pulse-1 at 3.4 MHz) working with rhodamine 6G. Intensity decay measurements were made by alternating collection of impulse and decays with the emission polarizer set at the magic angle position. The excitation pulse profiles were recorded slightly away from the excitation wavelength with a scattering suspension. For the decays, a cutoff filter was used to effectively remove excitation light. Emission light was passed through a depolarizer before reaching the monochromator (Jobin-Yvon HR320 with a 100 lines/mm grating) and detected using a Hamamatsu 2809U-01 microchannel plate photomultiplier. No less than 10,000 counts were accumulated at the maximum channel. The decay curves were analyzed using a nonlinear least squares reconvolution method [28].
different from those of PDIvis (Table 1, Fig. 2). The absorption and fluorescence emission maxima of PDInir are 150e200 nm red shifted, and the Stokes shift increases to 40e70 nm, relative to PDIvis. By encapsulating the dyes in silica nanoparticles, their absorption and emission spectra are not changed appreciably (Fig. 2(B)). Since s-SiNP-PDIvis have a much lower volume than lSiNP-PDIvis but the same PDIvis concentration, the emission per particle is also much smaller. However, for the same weight concentration of nanoparticles the emission intensity of the smaller nanoparticles is higher due to the reduced light scattering (Fig. 2(C)).
3. Results and discussion Silica nanoparticles with emission in the visible and NIR regions of the spectrum were obtained by encapsulation of the corresponding dyes (PDIvis or PDInir), using a modified Stöber method [25]. The two terminal triethoxysilyl groups, present in both dyes, were used as a secondary silica source to incorporate the dye molecules in the silica network. The size of the fluorescent nanoparticles was obtained by transmission electron microscopy (TEM, Fig. 1), with the average particle diameter recovered from TEM equal to (33 3) nm for s-SiNP-PDIvis, (3.3 0.3) 102 nm for lSiNP-PDIvis and (3.0 0.1) 102 nm for SiNP-PDInir (Fig. 1). The photophysical properties of PDInir, in particular the absorption and fluorescence emission spectra, are dramatically
Fig. 2. Normalized absorption spectra (A, B, dashed lines) and fluorescence spectra (A, B, solid lines, and C e measured in a fluorimeter) in 1,4-dioxane of (A) PDIvis and PDInir, (B) fluorescent nanoparticles l-SiNP-PDIvis and SiNP-PDInir, and (C) nanoparticles of different dimensions, s-SiNP-PDIvis (ca. 30 nm diameter, solid line) and lSiNP-PDIvis (ca. 300 nm diameter, dashed line). All spectra were recorded at 20 C.
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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fluorimeter (up to 850 nm while our microscope’s limit is 800 nm) and the lower sensitivity of the microscope PMT for wavelengths above 750 nm. To illustrate the application of l-SiNP-PDIvis and SiNP-PDInir as markers for LSCFM in vivo imaging, we internalized the particles in HEK293 cells, which were co-stained with a plasma membrane marker (AF594-WGA) and/or a nucleus marker (Hoechst 33258). LSCFM images show that both l-SiNP-PDIvis and SiNP-PDInir were efficiently internalized in the cells (Fig. 4). The major difference between the two sets of nanoparticles is that, while l-SiNP-PDIvis is preferably excited at 488 nm (argon laser), showing emission in the green region of the spectra, SiNPPDInir can be excited at 633 nm (using a low power/low cost HeNe laser) with the emission observed in the NIR. The use of markers emitting NIR light presents several advantages over lower wavelength light (visible) emitting dyes. In fact, not only NIR offers higher tissue penetration in biological specimens, [15] but also there is lower interference from sample autofluorescence in the NIR region, as well as less light scattering due to the l4 dependence of scattering intensity. Additionally, NIR dyes can be excited using inexpensive diode or He:Ne lasers in LSCFM imaging.
A
B
Normalized intensity (a.u.)
Nanoparticles cast from a dioxane dispersion into a glass slide were imaged by LSCFM, using the 488 nm line of an argon ion laser for l-SiNP-PDIvis (detection at 520e650 nm) and a He:Ne laser (633 nm) for SiNP-PDInir (detection at 680e800 nm). The images of l-SiNP-PDIvis and SiNP-PDInir (Fig. 3(A) and (C)) show that the nanoparticles can be individually identified in both cases (they have diameters approximately at the resolution limit of LSCFM). By comparing the images obtained by LSCFM (Fig. 3) and TEM (Fig. 1) we conclude that both dyes were successfully homogeneously incorporated in the silica nanoparticles to produce bright nanoparticles with low size dispersity. The LSCFM images can also be acquired sequentially, by varying the detection wavelength in 10 nm intervals, and from these we can reconstruct the emission spectrum of the individual nanoparticles, shown in Fig. 3(B) and (D) as an average over 500 nm diameter regions of the film centered over different nanoparticles. The fluorescence microspectra of the nanoparticles obtained by LSCFM closely follow those obtained in the fluorimeter for the nanoparticles in solution, with the loss in resolution justified by the larger bandgap (10 nm) necessary to measure the LSCFM spectra, and the difference observed in the red region of the spectra (>750 nm, Fig. 3(D)) caused by the larger detection limit of the
5
C
560
600
λ (nm)
670
640
D
Normalized intensity (a.u.)
520
710
750
790
λ (nm)
830
Fig. 3. LSCFM images (pseudo color) of (A) l-SiNP-PDIvis cast from ethanol dispersion onto a glass slide, imaged with the 488 nm laser line and (C) SiNP-PDInir cast from a dioxane dispersion onto a glass slide, imaged with the 633 nm laser line (HeNe). The fluorescence emission spectra of individual l-SiNP-PDIvis (B) and SiNP-PDInir (D) nanoparticles cast on glass slides and measured by LSCFM (thick lines) are similar to the spectra of the nanoparticles in dispersion, obtained by fluorescence spectroscopy (thin lines), with the difference above 750 nm being due to the lower sensitivity of the microscope PMT for wavelengths above 750 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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633 nm laser with 325 mW power (measured at the sample surface) and the images were collected sequentially (5.12 s per scan). The decrease in integrated fluorescence intensity presented in Fig. 5 shows that the intensity of DiIC18(5) decreases 50% after 4 scans and more than 95% after 20 scans, while SiNP-PDInir suffer only a decrease of 15% after 20 scans. One of the advantages of the NIR nanoparticles is that they do not emit in the same region as most of the dyes presently used for fluorescence imaging, including most fluorescent proteins. In fact, both the absorption and fluorescence bands of the SiNP-PDInir are well separated from those of the green fluorescent protein (GFP), with the wavelength range used for GFP imaging falling in the region of minimum absorption and no emission by SiNP-PDInir (Fig. 6). The ability for simultaneous independent in vivo four-color imaging of cells labeled with our NIR-emitting particles, GFP and two other dyes, was tested using HEK293 cells transfected with free GFP. We internalized SiNP-PDInir (with a non-optimized protocol) and further stained the cells with AF594-WGA plasma membrane marker and Hoechst 33258 nucleus marker. LSCFM images show that the nanoparticles were efficiently internalized by the cells and dispersed in the cytosol, being easily and clearly distinguishable from the GFP, the membrane and the nucleus markers (Fig. 7). In cell imaging applications, nanoparticles smaller than SiNPPDInir and l-SiNP-PDIvis (both around 300 nm diameter) are often preferred, because smaller particles can more easily access different
SiP-PDInir
The in vivo photostability of the labeled nanoparticles was compared with that of a commercial membrane marker, DiIC18(5), a Cy5 derivative with excitation maximum at 650 nm and emission maximum at 670 nm. This DiIC18(5) marker is widely used in LSCFM, and it has a Cy5 backbone showing fluorescence properties similar to those of PDInir. HEK293 cells were incubated with either SiNP-PDInir or DiIC18(5) in a non-optimized protocol. Due to the oxygen shielding effect of the silica shell, our nanoparticles show a remarkably higher photostability than the membrane marker during laser scanning. Both samples were scanned with a HeNe
Scan 10
Scan 20
Normalized fluorescence intensity
DiIC18(5)
Scan 1
1 0.8
0.6 0.4 0.2 0
1
5
9
13
17
number of scans
Fig. 4. (Top) LSCFM image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow) and Hoechst 33258 nucleus marker (blue), with internalized silica nanoparticles appearing in the cytosol (shown in red). SiNP-PDInir were excited at 633 nm (Top) and l-SiNP-PDIvis were excited at 488 nm (Bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fluorescence intensity evolution of SiNP-PDInir and DiIC18(5) membrane marker stained in HEK293 cells, excited with a HeNe 633 nm laser, over a set of 20 scans (5.12 s per scan). The error bars were determined for the standard deviation from different experiments. The fluorescence intensity was obtained by integrating the 645e790 nm wavelength range. The inset show confocal fluorescence images (image size 62.5 62.5 mm, each) of HEK293 cells loaded with SiNP-PDInir (upper row) and DiIC18(5) membrane marker (lower row) obtained at different scans. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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increase in the distribution homogeneity of the nanoparticles in the cytosol. The viability of the HEK293 cells upon internalization of the silica nanoparticles was evaluated through LDH leakage assays, after 18 h of incubation with different concentrations of nonfluorescent nanoparticles (to avoid cross talk between the PDI and the dye used in the viability test). We tested bare silica nanoparticles (s-SiNP) and the same nanoparticles after surface modification with RGD (s-SiNP-RGD). Control cells were cultured in particle-free media, and were run in parallel to treated groups. Cell viability of the control cells was set at 100%, and the values
Fig. 6. Normalized excitation (dashed curves) and emission (solid curves, measured in the fluorimeter) spectra of GFP in water (thin lines) and SiNP-PDInir nanoparticles in dioxane dispersion (thick lines). The SiNP-PDInir spectra do not overlap with GFP spectra, thus allowing simultaneous imaging of both dyes. All spectra were recorded at 20 C.
intracellular organelles. However, smaller nanoparticles also carry a lower amount of fluorescent molecules and thus are less bright than their larger counterparts. In order to test the performance of smaller fluorescent SiNPs for in vivo imaging, we prepared s-SiNPPDIvis (with ca. 30 nm diameter), which were internalized in HEK293 cells, co-stained with the AF594-WGA plasma membrane marker (Fig. 8 top). The nanoparticles not only are well dispersed in the cytosol, but also provide intense fluorescence emission under standard LSCFM experimental conditions. The s-SiNP-PDIvis nanoparticles were further surface modified with an argininee glycineeaspartic acid (RGD) oligopeptide, an integrin-binding motif known to promote cellular adhesion, with potential tumortargeting properties. Internalization of the resulting s-SiNP-PDIvis-RGD nanoparticles in HEK293 cells (Fig. 8 bottom) yielded an
Fig. 7. Confocal fluorescence image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow) and Hoechst 33258 nucleus marker (blue). GFP in transfected cells is shown in green. The SiNP-PDInir nanoparticles, shown in red, appear in endocytic vesicles (probably because of nanoparticles aggregation due to nonoptimized internalization) and dispersed in the cytosol (non-aggregated nanoparticles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. LSCFM image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow), showing internalized PDIvis labeled smaller silica nanoparticles (excited at 488 nm, shown in red) with very good brightness. Although the nonsurface-modified s-SiNP-PDIvis nanoparticles appear well dispersed in the cytosol (top), the s-SiNP-PDIvis-RGD featuring an arginineeglycineeaspartic acid (RGD) oligopeptide on the surface are even more homogeneously distributed in the cytosol (bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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R.S. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009; SFRH/ BD/89615/2012) and Pos-Doc (SFRH/BPD/71249/2010; SFRH/BPD/ 64320/2009) grants. The authors thank Dr. Aleksander Fedorov (CQFM-IN) for technical assistance with the picosecond lifetime decay measurements.
Cell Viability (%)
100 80 60
References
40 20 0 0
50
100
150
200
250
[Nanoparticles](μg/ml) Fig. 9. Viability of HEK293 cells after 18 h incubation with different concentrations of s-SiNP-RGD (open symbols) and s-SiNP (closed symbols). The viability was evaluated through LDH leakage assays. Control cells were cultured in particle-free media, and were run in parallel to treatment groups. Cell viability of the control cells was set at 100%, and the values obtained for cells exposed to the different nanoparticles are presented as percentages of the control.
obtained for cells exposed to the different nanoparticles are presented as percentages of the control, with a standard deviation calculated from three independent experiments. No change could be detected in cell viability for incubation with either bare or RGDmodified silica nanoparticles, indicating that no major toxicity issues are associated with the use of our nanoparticles for in vivo imaging (Fig. 9). 4. Conclusions We synthesized two PDI dyes modified with alkoxysilane groups for incorporation onto silica structures, one with emission in the visible (PDIvis with fluorescence quantum yield of 90%) and the other in the NIR (PDInir with fluorescence quantum yield of 24%, among the best for NIR dyes). The dyes were homogeneously incorporated into monodisperse silica nanoparticles with diameters of ca. 30e300 nm, yielding very brightly fluorescent silica nanoparticles. All particles, either bare or surface-modified with an arginineeglycineeaspartic acid (RGD) tumor targeting oligopeptide, were well internalized in HEK293 cells, with no effect on cell viability. The NIR emitting nanoparticles, SiNP-PDInir, can even be used in cells expressing high levels of fluorescent proteins and/or costained with different fluorescent dyes, both of which usually emit in the visible region of the spectrum. The NIR emitting nanoparticles are not only remarkably bright, but they also exhibit very high photostability compared to other currently used NIR markers. The possibility to produce both the NIR and the visible fluorescent silica nanoparticles in different sizes and their straightforward surface modification for better biocompatibility and/or for targeting to specific types of cells or organelles inside the cell, provide a very versatile and efficient platform for fluorescence microscopy bioimaging, in particular using laser scanning techniques, where several markers are often used simultaneously. Acknowledgments This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER), project PTDC/ CTM-NAN/2354/2012, and projects PEst-OE/CTM/LA0024/2013, RECI/QEQ-QIN/0189/2012 and RECI/CTM-POL/0342/2012. T.R., A.S.,
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Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging Tânia Ribeiro, Sebastian Raja, Ana S. Rodrigues, Fábio Fernandes, Carlos Baleizão*, José Paulo S. Farinha* CQFM e Centro de Química-Física Molecular and IN e Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2014 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online xxx
We describe the preparation of very bright and photostable fluorescent silica nanoparticles (SiNPs), with emission in the visible or in the near infrared (NIR). The nanoparticles can be prepared with diameters of 30e300 nm with low size dispersity, and incorporate perylenediimide (PDI) dyes modified with alkoxysilane groups that react with the silica structure. One of the dyes (PDIvis) emits in the green and the other (PDInir) in the NIR (with fluorescence quantum yields of 90% and 24%, respectively). Silica nanoparticles containing these dyes were efficiently internalized in HEK293 cells with low toxicity and reveled very high photostability, showing good potential for use as a platform for in vivo laser scanning bioimaging applications. The NIR emitting nanoparticles, in particular, can be used in multi-color imaging, even in cells expressing high levels of fluorescent proteins and/or co-stained with different fluorescent dyes (most of which commonly emit at wavelengths lower than the NIR). Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Fluorescent silica nanoparticles Perylenediimide NIR dyes Laser scanning confocal fluorescence microscopy Bioimaging RGD
1. Introduction Nanoparticle-based fluorescent imaging agents offer many advantages over molecular dyes, such as improved photo-robustness of the encapsulated dyes, the ability to carry large payloads of the optical probes, and the possibility of decorating the surface with specific groups. Additionally, nanoparticles can also carry and release drugs, leading to the development of different systems for use in combined diagnostic and therapeutic (theranostic) applications [1]. Silica nanoparticles (SiNPs), in particular, have been successfully used in nanomedicine, [2,3] as supports or carriers in drug delivery [4] and imaging [5]. The increasing interest in SiNPs is due to the possibility of tuning their diameter and porosity, functionalize the surface, load large amounts of fluorophores, drugs or other molecules, and control if and how the cargo is released [6]. Perylenediimide based dyes (PDIs) have many applications [7e 9] due to their typically high photochemical stability and extinction coefficients, and large fluorescence quantum yields [10]. The
* Corresponding authors. Tel.: þ351 218419221. E-mail addresses: [email protected] (C. Baleizão), farinha@ tecnico.ulisboa.pt (J.P.S. Farinha).
photophysical properties of PDIs can be modulated by introducing appropriate substituents in the imide group (to change solubility or allow immobilization) or in the perylene core (affecting the electronic and optical properties) [11]. By incorporating PDI dyes in SiNPs, one can obtain higher brightness, better photostability (due to lower oxygen concentration and diffusivity inside the nanoparticles) and also widen the range of solvents/materials in which they can be used by straightforward surface modification of the nanoparticles. We have synthesized two different PDI derivatives modified with alkoxysilane groups for incorporation into silica nanostructures (Scheme 1), one with emission in the green region of the spectrum (530e600 nm) and excitation at 450e530 nm (PDIvis), and the other with excitation in the red and emitting in the near infrared (NIR) region (PDInir) [12,13]. The dyes were incorporated in SiNPs (Scheme 2) with diameters at the limit of the optical resolution (ca. 300 nm, l-SiNP) and also in smaller nanoparticles (ca. 30 nm, s-SiNP), for testing in laser scanning confocal fluorescence microscopy (LSCFM) bioimaging. Due to the increased brightness and photostability of the nanoparticles relative to the isolated dyes, their biocompatibility (unaffected cell viability) and efficient cell internalization, these nanoparticles are very promising for in vivo laser-scanning imaging applications.
http://dx.doi.org/10.1016/j.dyepig.2014.03.026 0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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2. Materials and methods 2.1. Materials
Scheme 1. Structure of the perylenediimide (PDI) derivatives, one with emission in the visible region of the spectrum (PDIvis) and the other emitting in the NIR (PDInir).
Absolute ethanol (Panreac, 99.5%), ammonium hydroxide solution 25% (Fluka), tetraethyl orthosilicate (TEOS, Aldrich, 98%), N-(3dimethylaminopropyl)-N0 -ethylcarbodiimide (EDC, Aldrich, 97%), (3-aminopropyl)trietoxysilane (APTES, Aldrich, 98%) and a RGD oligopeptide (Gly-Arg-Gly-Asp-Ser-Pro, Aldrich) were used as received. Commercial toluene and dimethyl sulfoxide (DMSO) were distilled over calcium hydride before use. Deionized water from a Millipore system Milli-Q 18 MU cm was used for fluorescence measurements and endocytosis experiments. Perylenediimide derivatives, PDIvis and PDInir were synthesized according to the literature [11,12]. Silica nanoparticles doped with both PDI derivatives were synthesized by the Stöber method [26]. Wheat germ agglutinin (WGA) coupled to Alexa Fluor (AF)-594, Hoechst 33258 and DiIC18(5) were purchased from Invitrogen (Carlsbad, CA). 2.2. Synthesis of the fluorescent silica nanoparticles
Furthermore, the possibility of using near infrared (NIR) light (750e1400 nm) instead of lower wavelength (visible) light presents several advantages for optical bioimaging applications [14]. In the NIR region there is less light scattering than in the visible, light absorption and autofluorescence by biological tissues is lower [15] and therefore NIR light has higher tissue penetration [16]. Also, NIR dyes can be excited using less expensive lasers (He:Ne or red diode lasers) and can be used in systems labeled with multiple dyes, since most of the dyes presently used for fluorescence imaging (including most fluorescent proteins) emit at lower wavelength, in the visible region [17]. In spite of the advantages of using NIR dyes for fluorescence bio-imaging and of encapsulating fluorescence dyes in silica nanoparticles, only a few examples of functionalized NIR dyes for incorporation into silica structures, mostly based in carbocyanides, have been described [18e20]. The diameter of the nanoparticles can easily be tuned during synthesis and their surface can be modified by standard procedures for better biocompatibility and/or to achieve targeting to specific types of cells or organelles inside the cell [21,22]. Those strategies include the incorporation of polymer chains (either through surface initiated polymerization or by covalent immobilization of chains), charged species (via covalent attachment), biomacromolecules (as peptides, DNA, antigens), etc. Here, we tested the surface modification of the nanoparticles with an arginineeglycineeaspartic acid (RGD) oligopeptide, an integrin-binding motif known to promote cellular adhesion and proliferation [23]. Since some integrins are specifically expressed in tumor endothelia and certain tumor cells, RGDs can provide tumor-homing properties [24]. Therefore, by decorating the SiNPs surface with a RGD motif, the particles are expected to show enhanced specificity in delivering anticancer drugs and contrast agents for cancer diagnosis and therapy [25].
Water, absolute ethanol, and ammonia solution were mixed in a plastic flask, and the mixture stirred at 30 C. TEOS and PDI were dissolved in absolute ethanol and added at the same time to this mixture. The reaction was kept for 24 h at 30 C with magnetic stirring, after which the nanoparticles were centrifuged at 10,000 rpm for 3 cycles of 30 min, and either dispersed in ethanol or dried in vacuum. The volumes (in mL) of ammonia:TEOS:ethanol:water used were: 1.5:4.5:110:9 and 30 mg of PDIvis for the smaller visible light-emitting nanoparticles s-SiNP-PDIvis; 7.6:3.1:100:13 and 20 mg of PDIvis for large visible light-emitting nanoparticles l-SiNP-PDIvis; and 2.5:1:33:6 and 2.2 mg of PDInir for the NIR-emitting nanoparticles SiNP-PDInir. 2.3. Surface modification of the smaller silica nanoparticles with a RGD oligopeptide Fluorescent and non-fluorescent silica nanoparticles (0.040 g sSiNP-PDIvis or s-SiNP, the later obtained with the same procedure but without using PDIvis) were surface functionalized with APTES (93 mL, 0.4 mmol) in toluene (2 mL) and the reaction was kept under reflux with a magnetic stirring for 24 h at 125 C. The nanoparticles were then recovered by centrifugation at 15,000 rpm (20 min), washed three times with ethanol and dried over night at 40 C. The resulting nanoparticles, s-SiNP-PDIvis-APTES and s-SiNP-APTES (0.033 g) were then mixed with RGD oligopeptide (0.0033 mmol), EDC (2 mL, 0.012 mmol) in distilled DMSO (1 mL) and the mixture stirred at 25 C for 24 h. The mixture was centrifuged at 15,000 rpm (20 min), washed three times with ethanol and the recovered products, s-SiNP-PDIvis-RGD and s-SiNP-RGD were dried over night at 40 C.
Scheme 2. Incorporation of PDI derivatives in silica nanoparticles (SiNPs) by covalent attachment to the silica network during synthesis by a modified Stöber method.
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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3
D
B
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Relative frequency
A 0,4
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0
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E
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0
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F
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Fig. 1. TEM images of s-SiNP-PDIvis (A), l-SiNP-PDIvis (B) and SiNP-PDInir (C), with corresponding diameter distributions (D, E and F, respectively). Scale bars 200 nm.
2.4. In vivo tests HEK293 cells were cultured in an atmosphere of 5% CO2/95% air at 37 C. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY) was used. Transfection was performed in serum-free medium with pEGFP-N1 (Clontech, Palo Alto, CA), and 24 h after cells were seeded in 8 well ibidi m-slides (Martinsried, Germany). Gencarrier-2 from Epoch Biolabs (Sugar Land, TX, USA) was used as the transfection reagent. Transfection medium was removed after 6 h and
Table 1 Photophysical parameters of the dyes PDIvis and PDInir and the fluorescent nanoparticles SiNP-PDIvis and SiNP-PDInir in 1,4-dioxane.
PDIvis SiNP-PDIvisc PDInir SiNP-PDInir a b c
labs max
lemi max
(nm)
(nm)
521 e 685 e
530 528 741 734
3
(M1cm1)a
56,162 e 41,700 e
FF
Brightnessb (104 M1cm1)
s (ns)
0.90 e 0.24 e
5.05 e 1.00 e
4.3 4.4 4.0 3.8
At the absorption maxima. The product between the absorptivity and the quantum yield. Values obtained for s-SiNP-PDIvis and l-SiNP-PDIvis.
cells were incubated with nanoparticles (250 mg/mL) in complete medium for another 12 h. Immediately before imaging, cells were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (AF594-WGA) (5 mg/mL in PBS), Hoechst 33258 (2 mg/mL in PBS) or DiIC18(5) (5 mM in PBS) at 37 C for 10e20 min. Excess dye was removed by washing 3 times with culture medium. 2.5. Cell viability tests Cell viability in the presence of different concentrations of nanoparticles was assessed through the lactate dehydrogenase (LDH) activity in extracellular medium. LDH activity was measured with the Citotoxicity Detection Kit Plus from Roche (Mannhein, Germany) on a BMG POLARstar Optima plate reader (BMG Labtechnologies, Offenburg, Germany). 2.6. Methods Laser scanning confocal fluorescence microscopy (LSCFM) images were obtained on a Leica TCS SP5 laser scanning microscope (Leica Mycrosystems CMS GmbH, Mannheim, Germany) using an inverted microscope (DMI6000) and a HCX PL APO CS 1.20 W 63.3 waterimmersion apochromatic objective (63.3 magnification and 1.2
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numerical aperture). Imaging and image spectral analysis used either the 488 nm line of an argon ion laser (for PDIvis, 21.4 mW at the sample surface) or the 633 nm line of a He:Ne laser (for PDInir, 325 mW at the sample surface). The laser light intensity was controlled by an acoustic-optical filter system. The laser power at the sample was measured with a PM100D optical power meter with an S120C sensor (Thorlabs, NJ). The fluorescence emission was collected at selected wavelengths, using the tunable system and beam splitter of the Leica TCS SPC5. The laser power and photomultiplier tube gain were constant for all measurements. The offset was chosen such that the photon counts outside the sample were negligible. The pinhole was always set at 1 Airy unit to discriminate stray light from out-of-focus plans. Images obtained at a resolution of 512 512 pixels were processed with the software ImageJ (http://rsbweb.nih.gov/ij/). Emission spectra were obtained in 500 nm regions of the images, with 10 nm steps and a bandwidth of 10 nm. The data was processed using the Leica Application SuiteAdvanced Fluorescence software to obtain the spectra in different regions with a 500 nm diameter in each image. TEM images were obtained on a Hitachi transmission electron microscope (Model H-8100 with a LaB6 filament) with an accelerator voltage of 200 kV. One drop of the nanoparticles dispersion in ethanol was placed on a carbon grid and dried in air before observation. The absorption spectra were recorded on a Shimadzu UV3101PC UV-vis-NIR spectrophotometer, and the fluorescence measurements were obtained on a Horiba Jobin Yvon Fluorolog 322 spectrofluorometer. For the quantum yield, Rhodamine B in ethanol was used as standard [27]. The fluorescence emission spectra were corrected to the photomultiplier response. Timeresolved picosecond fluorescence intensity decays were obtained by the single-photon timing method in a setup consisting of a diode-pumped solid state Nd:YVO4 laser (Milennia Xs, Spectra Physics) synchronously pumping a mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, with tuning range 700e1000 nm, output pulses of 100 fs, and 80 MHz repetition rate that can be reduced to 4 MHz by a pulse picker) or a cavity dumped dye laser (701-2, Coherent, delivering 3e4 ps pulses of ca. 40 nJ pulse-1 at 3.4 MHz) working with rhodamine 6G. Intensity decay measurements were made by alternating collection of impulse and decays with the emission polarizer set at the magic angle position. The excitation pulse profiles were recorded slightly away from the excitation wavelength with a scattering suspension. For the decays, a cutoff filter was used to effectively remove excitation light. Emission light was passed through a depolarizer before reaching the monochromator (Jobin-Yvon HR320 with a 100 lines/mm grating) and detected using a Hamamatsu 2809U-01 microchannel plate photomultiplier. No less than 10,000 counts were accumulated at the maximum channel. The decay curves were analyzed using a nonlinear least squares reconvolution method [28].
different from those of PDIvis (Table 1, Fig. 2). The absorption and fluorescence emission maxima of PDInir are 150e200 nm red shifted, and the Stokes shift increases to 40e70 nm, relative to PDIvis. By encapsulating the dyes in silica nanoparticles, their absorption and emission spectra are not changed appreciably (Fig. 2(B)). Since s-SiNP-PDIvis have a much lower volume than lSiNP-PDIvis but the same PDIvis concentration, the emission per particle is also much smaller. However, for the same weight concentration of nanoparticles the emission intensity of the smaller nanoparticles is higher due to the reduced light scattering (Fig. 2(C)).
3. Results and discussion Silica nanoparticles with emission in the visible and NIR regions of the spectrum were obtained by encapsulation of the corresponding dyes (PDIvis or PDInir), using a modified Stöber method [25]. The two terminal triethoxysilyl groups, present in both dyes, were used as a secondary silica source to incorporate the dye molecules in the silica network. The size of the fluorescent nanoparticles was obtained by transmission electron microscopy (TEM, Fig. 1), with the average particle diameter recovered from TEM equal to (33 3) nm for s-SiNP-PDIvis, (3.3 0.3) 102 nm for lSiNP-PDIvis and (3.0 0.1) 102 nm for SiNP-PDInir (Fig. 1). The photophysical properties of PDInir, in particular the absorption and fluorescence emission spectra, are dramatically
Fig. 2. Normalized absorption spectra (A, B, dashed lines) and fluorescence spectra (A, B, solid lines, and C e measured in a fluorimeter) in 1,4-dioxane of (A) PDIvis and PDInir, (B) fluorescent nanoparticles l-SiNP-PDIvis and SiNP-PDInir, and (C) nanoparticles of different dimensions, s-SiNP-PDIvis (ca. 30 nm diameter, solid line) and lSiNP-PDIvis (ca. 300 nm diameter, dashed line). All spectra were recorded at 20 C.
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fluorimeter (up to 850 nm while our microscope’s limit is 800 nm) and the lower sensitivity of the microscope PMT for wavelengths above 750 nm. To illustrate the application of l-SiNP-PDIvis and SiNP-PDInir as markers for LSCFM in vivo imaging, we internalized the particles in HEK293 cells, which were co-stained with a plasma membrane marker (AF594-WGA) and/or a nucleus marker (Hoechst 33258). LSCFM images show that both l-SiNP-PDIvis and SiNP-PDInir were efficiently internalized in the cells (Fig. 4). The major difference between the two sets of nanoparticles is that, while l-SiNP-PDIvis is preferably excited at 488 nm (argon laser), showing emission in the green region of the spectra, SiNPPDInir can be excited at 633 nm (using a low power/low cost HeNe laser) with the emission observed in the NIR. The use of markers emitting NIR light presents several advantages over lower wavelength light (visible) emitting dyes. In fact, not only NIR offers higher tissue penetration in biological specimens, [15] but also there is lower interference from sample autofluorescence in the NIR region, as well as less light scattering due to the l4 dependence of scattering intensity. Additionally, NIR dyes can be excited using inexpensive diode or He:Ne lasers in LSCFM imaging.
A
B
Normalized intensity (a.u.)
Nanoparticles cast from a dioxane dispersion into a glass slide were imaged by LSCFM, using the 488 nm line of an argon ion laser for l-SiNP-PDIvis (detection at 520e650 nm) and a He:Ne laser (633 nm) for SiNP-PDInir (detection at 680e800 nm). The images of l-SiNP-PDIvis and SiNP-PDInir (Fig. 3(A) and (C)) show that the nanoparticles can be individually identified in both cases (they have diameters approximately at the resolution limit of LSCFM). By comparing the images obtained by LSCFM (Fig. 3) and TEM (Fig. 1) we conclude that both dyes were successfully homogeneously incorporated in the silica nanoparticles to produce bright nanoparticles with low size dispersity. The LSCFM images can also be acquired sequentially, by varying the detection wavelength in 10 nm intervals, and from these we can reconstruct the emission spectrum of the individual nanoparticles, shown in Fig. 3(B) and (D) as an average over 500 nm diameter regions of the film centered over different nanoparticles. The fluorescence microspectra of the nanoparticles obtained by LSCFM closely follow those obtained in the fluorimeter for the nanoparticles in solution, with the loss in resolution justified by the larger bandgap (10 nm) necessary to measure the LSCFM spectra, and the difference observed in the red region of the spectra (>750 nm, Fig. 3(D)) caused by the larger detection limit of the
5
C
560
600
λ (nm)
670
640
D
Normalized intensity (a.u.)
520
710
750
790
λ (nm)
830
Fig. 3. LSCFM images (pseudo color) of (A) l-SiNP-PDIvis cast from ethanol dispersion onto a glass slide, imaged with the 488 nm laser line and (C) SiNP-PDInir cast from a dioxane dispersion onto a glass slide, imaged with the 633 nm laser line (HeNe). The fluorescence emission spectra of individual l-SiNP-PDIvis (B) and SiNP-PDInir (D) nanoparticles cast on glass slides and measured by LSCFM (thick lines) are similar to the spectra of the nanoparticles in dispersion, obtained by fluorescence spectroscopy (thin lines), with the difference above 750 nm being due to the lower sensitivity of the microscope PMT for wavelengths above 750 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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633 nm laser with 325 mW power (measured at the sample surface) and the images were collected sequentially (5.12 s per scan). The decrease in integrated fluorescence intensity presented in Fig. 5 shows that the intensity of DiIC18(5) decreases 50% after 4 scans and more than 95% after 20 scans, while SiNP-PDInir suffer only a decrease of 15% after 20 scans. One of the advantages of the NIR nanoparticles is that they do not emit in the same region as most of the dyes presently used for fluorescence imaging, including most fluorescent proteins. In fact, both the absorption and fluorescence bands of the SiNP-PDInir are well separated from those of the green fluorescent protein (GFP), with the wavelength range used for GFP imaging falling in the region of minimum absorption and no emission by SiNP-PDInir (Fig. 6). The ability for simultaneous independent in vivo four-color imaging of cells labeled with our NIR-emitting particles, GFP and two other dyes, was tested using HEK293 cells transfected with free GFP. We internalized SiNP-PDInir (with a non-optimized protocol) and further stained the cells with AF594-WGA plasma membrane marker and Hoechst 33258 nucleus marker. LSCFM images show that the nanoparticles were efficiently internalized by the cells and dispersed in the cytosol, being easily and clearly distinguishable from the GFP, the membrane and the nucleus markers (Fig. 7). In cell imaging applications, nanoparticles smaller than SiNPPDInir and l-SiNP-PDIvis (both around 300 nm diameter) are often preferred, because smaller particles can more easily access different
SiP-PDInir
The in vivo photostability of the labeled nanoparticles was compared with that of a commercial membrane marker, DiIC18(5), a Cy5 derivative with excitation maximum at 650 nm and emission maximum at 670 nm. This DiIC18(5) marker is widely used in LSCFM, and it has a Cy5 backbone showing fluorescence properties similar to those of PDInir. HEK293 cells were incubated with either SiNP-PDInir or DiIC18(5) in a non-optimized protocol. Due to the oxygen shielding effect of the silica shell, our nanoparticles show a remarkably higher photostability than the membrane marker during laser scanning. Both samples were scanned with a HeNe
Scan 10
Scan 20
Normalized fluorescence intensity
DiIC18(5)
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1 0.8
0.6 0.4 0.2 0
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Fig. 4. (Top) LSCFM image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow) and Hoechst 33258 nucleus marker (blue), with internalized silica nanoparticles appearing in the cytosol (shown in red). SiNP-PDInir were excited at 633 nm (Top) and l-SiNP-PDIvis were excited at 488 nm (Bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fluorescence intensity evolution of SiNP-PDInir and DiIC18(5) membrane marker stained in HEK293 cells, excited with a HeNe 633 nm laser, over a set of 20 scans (5.12 s per scan). The error bars were determined for the standard deviation from different experiments. The fluorescence intensity was obtained by integrating the 645e790 nm wavelength range. The inset show confocal fluorescence images (image size 62.5 62.5 mm, each) of HEK293 cells loaded with SiNP-PDInir (upper row) and DiIC18(5) membrane marker (lower row) obtained at different scans. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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increase in the distribution homogeneity of the nanoparticles in the cytosol. The viability of the HEK293 cells upon internalization of the silica nanoparticles was evaluated through LDH leakage assays, after 18 h of incubation with different concentrations of nonfluorescent nanoparticles (to avoid cross talk between the PDI and the dye used in the viability test). We tested bare silica nanoparticles (s-SiNP) and the same nanoparticles after surface modification with RGD (s-SiNP-RGD). Control cells were cultured in particle-free media, and were run in parallel to treated groups. Cell viability of the control cells was set at 100%, and the values
Fig. 6. Normalized excitation (dashed curves) and emission (solid curves, measured in the fluorimeter) spectra of GFP in water (thin lines) and SiNP-PDInir nanoparticles in dioxane dispersion (thick lines). The SiNP-PDInir spectra do not overlap with GFP spectra, thus allowing simultaneous imaging of both dyes. All spectra were recorded at 20 C.
intracellular organelles. However, smaller nanoparticles also carry a lower amount of fluorescent molecules and thus are less bright than their larger counterparts. In order to test the performance of smaller fluorescent SiNPs for in vivo imaging, we prepared s-SiNPPDIvis (with ca. 30 nm diameter), which were internalized in HEK293 cells, co-stained with the AF594-WGA plasma membrane marker (Fig. 8 top). The nanoparticles not only are well dispersed in the cytosol, but also provide intense fluorescence emission under standard LSCFM experimental conditions. The s-SiNP-PDIvis nanoparticles were further surface modified with an argininee glycineeaspartic acid (RGD) oligopeptide, an integrin-binding motif known to promote cellular adhesion, with potential tumortargeting properties. Internalization of the resulting s-SiNP-PDIvis-RGD nanoparticles in HEK293 cells (Fig. 8 bottom) yielded an
Fig. 7. Confocal fluorescence image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow) and Hoechst 33258 nucleus marker (blue). GFP in transfected cells is shown in green. The SiNP-PDInir nanoparticles, shown in red, appear in endocytic vesicles (probably because of nanoparticles aggregation due to nonoptimized internalization) and dispersed in the cytosol (non-aggregated nanoparticles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. LSCFM image of HEK293 cells stained with AF594-WGA plasma membrane marker (yellow), showing internalized PDIvis labeled smaller silica nanoparticles (excited at 488 nm, shown in red) with very good brightness. Although the nonsurface-modified s-SiNP-PDIvis nanoparticles appear well dispersed in the cytosol (top), the s-SiNP-PDIvis-RGD featuring an arginineeglycineeaspartic acid (RGD) oligopeptide on the surface are even more homogeneously distributed in the cytosol (bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026
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R.S. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009; SFRH/ BD/89615/2012) and Pos-Doc (SFRH/BPD/71249/2010; SFRH/BPD/ 64320/2009) grants. The authors thank Dr. Aleksander Fedorov (CQFM-IN) for technical assistance with the picosecond lifetime decay measurements.
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[Nanoparticles](μg/ml) Fig. 9. Viability of HEK293 cells after 18 h incubation with different concentrations of s-SiNP-RGD (open symbols) and s-SiNP (closed symbols). The viability was evaluated through LDH leakage assays. Control cells were cultured in particle-free media, and were run in parallel to treatment groups. Cell viability of the control cells was set at 100%, and the values obtained for cells exposed to the different nanoparticles are presented as percentages of the control.
obtained for cells exposed to the different nanoparticles are presented as percentages of the control, with a standard deviation calculated from three independent experiments. No change could be detected in cell viability for incubation with either bare or RGDmodified silica nanoparticles, indicating that no major toxicity issues are associated with the use of our nanoparticles for in vivo imaging (Fig. 9). 4. Conclusions We synthesized two PDI dyes modified with alkoxysilane groups for incorporation onto silica structures, one with emission in the visible (PDIvis with fluorescence quantum yield of 90%) and the other in the NIR (PDInir with fluorescence quantum yield of 24%, among the best for NIR dyes). The dyes were homogeneously incorporated into monodisperse silica nanoparticles with diameters of ca. 30e300 nm, yielding very brightly fluorescent silica nanoparticles. All particles, either bare or surface-modified with an arginineeglycineeaspartic acid (RGD) tumor targeting oligopeptide, were well internalized in HEK293 cells, with no effect on cell viability. The NIR emitting nanoparticles, SiNP-PDInir, can even be used in cells expressing high levels of fluorescent proteins and/or costained with different fluorescent dyes, both of which usually emit in the visible region of the spectrum. The NIR emitting nanoparticles are not only remarkably bright, but they also exhibit very high photostability compared to other currently used NIR markers. The possibility to produce both the NIR and the visible fluorescent silica nanoparticles in different sizes and their straightforward surface modification for better biocompatibility and/or for targeting to specific types of cells or organelles inside the cell, provide a very versatile and efficient platform for fluorescence microscopy bioimaging, in particular using laser scanning techniques, where several markers are often used simultaneously. Acknowledgments This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER), project PTDC/ CTM-NAN/2354/2012, and projects PEst-OE/CTM/LA0024/2013, RECI/QEQ-QIN/0189/2012 and RECI/CTM-POL/0342/2012. T.R., A.S.,
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Please cite this article in press as: Ribeiro T, et al., NIR and visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.026