Facile Preparation of Water Dispersible Red Fluorescent Organic Nanoparticles for Cell Imaging

Abstract

Red fluorescent organic nanopaticles (FONs) based on a diarylacrylonitrile derivative conjugated molecule were facilely prepared by surfactant modification. Such red FONs showed excellent water solubility and biocompatibility, making them promising for cell imaging applications.

Introduction

Optical bioimaging is one of the rapid growing areas in biological and biomedical research.1 Fluorescence cellular probes with red/near-infrared (R/NIR, > 600 nm) emission are highly desirable for biological applications due to their low optical absorption and autofluorescence of biological media.2-4 Up to date, various fluorescent materials including organic dyes,5-7 fluorescent proteins,8 fluorescent inorganic nanoparticles (NPs) 9-14 have been used as R/NIR probes. However, many of these fluorescent materials often suffered from obvious disadvantages such as water insolubility, photo-bleaching and toxicity, which severely limited their practical bioimaging applications. For example, most of organic dyes are intrinsic hydrophobic and instable in biological media.15 Usage of fluorescent proteins is often limited due to the high cost, low molar absorptivity and low photobleaching thresholds,16 while inorganic NPs or quantum dots are non-biodegradable and often toxic to living organisms.17-19 Com-pared with the fluorescent bioprobes currently available, organic nanoparticles (FONs) are receiving more and more attentions due to flexible synthetic approaches of these small organic molecules and their biodegradation potential.20-24 Because of these virtues, various FONs including fluorescent conjugated polymers, polydopamine nanoparticles and aggre-gation- induced-emission (AIE), or aggregation induced emi-ssion enhancement (AIEE) materials have been reported in recent years.25-31 Among them, the AIE or AIEE based FONs have attracted great research interest because they could overcome the notorious aggregation-caused quenching (ACQ) effect of most organic dyes. Different AIE or AIEE units such as siloles,32-34 cyano-substituted diarylethene,35-39 tetraphenylethene, 40-43 triphenylethene,44-48 distyrylanthracene derivatives49-53 conjugated molecules have been examined for chemosensors and bioimaging applications.

However, direct employment of AIE or AIEE based FONs for bioimaging has been proven problematic due to the strong hydrophobicity of most of AIE or AIEE units. The introduction of charges into AIE (or AIEE) materials may improve their solubility in aqueous media, but the electric charges of the highly concentrated ionized dyes would affect intracellular physiology and sometimes even kill the cells.54 Therefore, facile preparation of novel FONs which exhibited enhanced water dispersibility, excellent biocompatibility and photoluminescent properties simultaneously is of great significance.

Scheme 1.The formation of hydrophilic red FONs.

In this connection, it has to be noted that Zhang et al. had earlier reported facile transformation of hydrophobic AIE (An18) into hydrophilic FONs by mixing the unit with a commercial surfactant pluronic F127.55 The FONs thus formed reveal not only high water solubility but also ex-cellent biocompatibility-a promising observation for cell imaging applications. One drawback, however, of these AIE-based FONs for bioimaging lies in the fact that their emission maxima fall in the range 530-550 nm, where inter-ference with the body optical absorption, light scattering, and autofluorescence of biological media is unavoidable.

Herein, we report a facile method for the preparation of hydrophilic red FONs based on a diarylacrylonitrile derivative (R-NH2) by surfactant modification (Scheme 1). The hydro-phobic R-NH2 was changed to hydrophilic by surrounding with F127 to form R-NH2-F127 FONs, which exhibit good water dispersibility and anti-ACQ property. To exploit the potential biomedical applications of such red FONs (R-NH2- F127), their biocompatibility as well as cell imaging applications were further investigated.

 

Experimental

Materials and Measurements. Thiophene, 3-(bromometh-yl)heptane, thiophene-2-carboxylic acid, thionyl chloride, dimethylamine, n-butyllithium, stannic chloride, 2-(4-amino-phenyl) acetonitrile, tetrabutylammonium hydroxide (0.8 M in methanol) purchased from Alfa Aesar were used as received. All other agents and solvents purchased from commercial sources were used directly without further purification. The synthetic route of R-NH2 was showed in Scheme 2, which was prepared according to the previous literature method.56 Its structure was characterized and confirmed by standard spectroscopic methods.

Scheme 2.Synthetic route of R-NH2.

Fluorescence spectra were measured on a PE LS-55 spectrometer with a slit width of 3 nm for both excitation and emission. Transmission electron microscopy (TEM) images were recorded on a JEM-1200EX microscope operated at 100 kV, the TEM specimens were made by placing a drop of the nanoparticles suspension on a carbon-coated copper grid. The FT-IR spectra were obtained in a transmission mode on a Perkin-Elmer Spectrum 100 spectrometer (Waltham, MA, USA). Typically, 8 scans at a resolution of 1 cm−1 were accumulated to obtain one spectrum. The size distributions measurement of the FONs in phosphate buffer solution (PBS) were determined using a zeta Plus apparatus (ZetaPlus, Brookhaven Instruments, Holtsville, NY).

Preparation of R-NH2-F127 FONs. The preparation of R-NH2-F127 FONs was carried out as follows. Approxi-mately 5 mg of synthesized dyes (R-NH2) was dissolved in 20 mL of THF and then added dropwise into the solution of Pluronic F127 (20 mg) in 20 mL of H2O in a 100 mL vial under sonication. And then the mixture was evaporated to remove the organic agent (THF) completely on a rotary evaporator at 40°C. To remove the excess Pluronic F127, the R-NH2-F127 water dispersion was treated by repeated centrifugal washing process for three times.

Cytotoxicity of R-NH2-F127 FONs. Cell morphology was observed to examine the effects of R-NH2-F127 FONs to A549 cells. Briefly, cells were seeded in 6-well micro-plates at a density of 1 × 105 cells mL–1 in 2 mL of respective media containing 10% FBS. After cell attachment, the plates were washed with PBS and the cells were treated with com-plete cell culture medium, or different concentrations of fluoridated R-NH2-F127 FONs prepared in 10% FBS con-taining media for 24 h. Then all samples were washed with PBS three times to remove the uninternalized FONs. The morphology of cells was observed by using an optical microscopy (Leica, Germany), the overall magnification was × 100.

The cell viability of R-NH2-F127 FONs on A549 cells was evaluated by cell counting kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-well microplates at a density of 5 × 104 cells mL–1 in 160 μL of respective media containing 10% FBS. After cell attachment for 24 h, the cells were incubated with 10, 20, 40, 80, 120 μg mL–1 R-NH2-F127 FONs for 8 and 24 h, respectively. Then nanoparticles were removed and cells were washed with PBS for three times. 10 μL of CCK-8 dye and 100 μL of DMEM cell culture medium were added to each well and incubated for 2 h at 37°C. Plates were then analyzed with a microplate reader (VictorШ, Perkin-Elmer). Measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The values were proportional to the number of live cells. The percent reduction of CCK-8 dye was compared to controls (cells not exposed to R-NH2-F127 FONs), which represented 100% CCK-8 reduction. Three replicate wells were used per microplate, and the experiment was repeated for three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD).

Confocal Microscopic Imaging of Cells using R-NH2-F127 FONs. A549 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U mL–1 penicillin, and 100 μg mL–1 of streptomycin. Cell culture was maintained at 37°C in a humidified condition of 95% air and 5% CO2 in culture medium. Culture medium was changed every three days for maintaining the exponential growth of the cells. On the day prior to treatment, cells were seeded in a glass bottom dish with a density of 1 × 105 cells per dish. On the day of treatment, the cells were incubated with R-NH2-F127 FONs at a final concentration of 100 μg mL–1 for 3 h at 37°C. Afterwards, the cells were washed for three times with PBS to remove the R-NH2-F127 FONs and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were taken with a Laser Scanning Confocal Microscope (LCSM) Zesis 710 3-channel (Zesis, Germany) with the excitation wavelengths of 543 nm.

 

Results and Discussion

The fluorescent dye R-NH2 is hydrophobic and emits strong crimson fluorescence in solid state. When R-NH2 is dissolved in THF, it has strong orange fluorescence in dispersed state. After modification by surfactant Pluronic F127, R-NH2 could be dispersed well in water via hydro-pholic interaction with F127, meanwhile, the fluorogen was still in aggregated state in the R-NH2-F127 nanoparticles, and the R-NH2-F127 composite emitted strong red fluore-scence in water with almost the same fluorescent intensity like that in pure THF solution (Fig. 1(a) and 1(b)), which show obvious anti-ACQ property. When the nanoparticles formed, the fluorescent wavelength of the fluorescence dye emerged a 22 nm red-shifted emission. The picture of preci-pitated R-NH2 in water was showed in Fig. 1(b) (right), which demonstrated similar emission wavelength like R-NH2- F127. The observed spectral shift is attributed presum-ably to the torsion-locking planarization of the distorted solution geometry of R-NH2 by aggregation, which narrows the optical bandgap with broadening π-electron conjugation. The appearance of longer-wavelength emission also sug-gested that close stacking of the planarized R-NH2 molecules was possibly accompanied with fluorescence-enhancing J-type π-aggregation, often occurring in diarylacrylonitrile derivatives.23,26 As shown in the inset of Figure 1(a), after surface modification with Pluronic F127, the surfactant modificated nanoparticles R-NH2-F127 were readily dis-persed in water, suggesting the successful formation of hydrophilic FONs.

Figure 1.(a) Fluorescence spectra of R-NH2 in THF and surfactant modificated nanoparticles (R-NH2-F127) dispersed in water. Inset: fluorescent image of R-NH2 in THF and R-NH2-F127 in water taken at 365 nm UV light; (b) Image of R-NH2 (left) in THF, R-NH2- F127 (middle) and R-NH2 (right) in water taken at visible light. (c) TEM image of R-NH2-F127 FONs dispersed in water, scale bar = 300 nm.

FT-IR spectra showed that a peak centered at 1102 cm–1, which is corresponding to the strecth vibration of C-O, was significantly enhanced in R-NH2-F127 FONs, demonstrat-ing the successful modification of R-NH2 dyes with Pluronic F127 (Fig. 2). The transmission electron microscopy (TEM) images further confirmed the formation of FONs. Some small organic spheres with diameters ranging from 40 to 60 nm, which derived from the assembly of R-NH2 and Pluronic F127 were observed (Fig. 1(c)). The size distribution of R-NH2- F127 FONs in PBS was tested using a zeta Plus particle size analyzer, showed the size distribution was 116.1 ± 6.9 nm, with a polydispersity index (PDI) of 0.254. The sizes characterized by TEM were somewhat smaller the zeta Plus particle size analyzer likely due to the shrinkage of micelle during the drying process. The formation of such nanostructures is likely due to the strong interactions between the alkyl chain of R-NH2 and the hydrophobic segments of F127, meanwhile, the hydrophilic segments of Pluronic F127 were covered on the spheres to render them water dis-persible. Taken together, excellent water solubility, small particles, biodegradable potential, and unique photolumine-scent (PL) properties (including broad excitation wavelenth and long emission wavelength (Fig. 3)) were found in the red FONs, so we expect that R-NH2-F127 FONs can be potential used for bioimaging applications.

Figure 2.FT-IR spectra of R-NH2, F127 and R-NH2-F127.

Figure 3.Excitation and emission spectra of R-NH2-F127 FONs.

Figure 4.Biocompatibility evaluation of R-NH2-F127 FONs. (a-c) optical microscopy images of A549 cells incubated with different concentrations of R-NH2-F127 FONs for 24 h, (a) control cells, (b) 40 μg mL–1, (c) 120 μg mL–1, (d) cell viability of R-NH2-F127 FONs with A549 cells for 8 and 24 h, respectively. Cell viability was determined by the WST assay

To test the potential biomedical applications of R-NH2-F127 FONs, their biocompatibility with A549 cells were subsquently examined.57-59 Figure 4(a)-(c) showed the micro-scopy images of cells when they were incubated with differ-ent concentrations of R-NH2-F127 FONs for 24 h, however, no significant cell morphology changes were observed. It could be seen that cells still attached very well to cell plate even the concentration of the FONs was up to 120 μg mL–1. The optical microscopy images implied good biocompati-bility of R-NH2-F127 FONs. Cell viability was further determined to quantitatively evaluate the biocompatibility of R-NH2-F127 FONs.60 As shown in Figure 4(d), no obvious cell viability decrease was found when cells were incubated with 40 μg mL–1 of R-NH2-F127 FONs for 8 and 24 h, respectively. The cell viability values were about 90% even the concentration was increased to 120 μg mL–1, further confirming the excellent biocompatibility of R-NH2-F127 FONs.

The cell imaging applications of R-NH2-F127 FONs were further explored. The confocal laser scanning microscope (CLSM) images of cells incubated with 100 μg mL–1of R-NH2- F127 FONs for 3 h were shown in Figure 5.61-63

As bright field images (Fig. 5(a)) indicated that cells still kept their normal morphologies, further evidencing the good biocompatibility of R-NH2-F127 FONs. When cells were excited with 543 nm laser, the cell uptake of R-NH2-F127 FONs could be clearly distinguished due to they were stain-ed by R-NH2-F127 FONs. Furthermore, many dark areas which were surrounded by R-NH2-F127 FONs areas also could be found from the CLSM images, which were likely the location of cell nuclues. Therefore, we could expect that the R-NH2-F127 FONs should be promising candidates for various biomedical applications due to the combined advant-ages for bio-applications, such as unique PL properties, good water solubility, excellent biocompatibility, and biodegradeble potential.

Figure 5.CLSM images of A549 cells incubated with 100 μg mL–1 of R-NH2-F127 FONs for 3 h. (a) bright field, (b) fluorescent image which were excited with 543 nm laser, (c) merged images. Scale bar = 20 μm.

 

Conclusion

In summary, a novel strategy for fabrication of red FONs (R-NH2-F127) was developed via mixing a hydrophobic diarylacrylonitrile derivative conjugated molecule (R-NH2) with a commercial surfactant Pluronic F127. These red FONs with diameter ranged from 40 to 60 nm could be facilely obtained and subsquently utilized for cell imaging applications. Our results demonstrated that such red FONs showed remarkable PL properties (anti-ACQ properties and broad excitation wavelength), excellent water solubility and biocompatibility, making them promising for bioimaging applications.