Quantum dots tagged poly(alkylcyanoacrylate) nanoparticles intended for bioimaging applications

Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Quantum dots tagged poly(alkylcyanoacrylate) nanoparticles intended for bioimaging applications Georgi Yordanov a , Margarita Simeonova b,c , Radostina Alexandrova d , Hideyuki Yoshimura e , Ceco Dushkin a,∗ a

Laboratory of Nanoparticle Science and Technology, Faculty of Chemistry, University of Sofia, 1164 Sofia, Bulgaria Department of Polymer Engineering, University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria Laboratory of Amphiphilic and Ionogenic Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bontchev Street, Block 103 A, 1113 Sofia, Bulgaria d Department of Oncovirology, Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Acad. G. Bontchev Street, Block 25, 1113 Sofia, Bulgaria e Department of Physics, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagava 214-8571, Japan b c

a r t i c l e

i n f o

Article history: Received 5 October 2008 Received in revised form 22 January 2009 Accepted 16 February 2009 Available online 25 February 2009 Keywords: CdSe CdS CdSe/CdS Core/shell Hybrid nanoparticles Nanocrystals Quantum dots Poly(butylcyanoacrylate) Fluorescence

a b s t r a c t Fluorescent quantum dots (QDs) tagged poly(butylcyanoacrylate) nanoparticles (PBCN) intended for cell fluorescent imaging were prepared and characterized. Highly fluorescent CdSe/CdS core-shell QDs were synthesized and successfully embedded into PBCN by interfacial polymerization in aqueous medium. Transmission electron microscopy, dynamic light scattering, and zeta potential measurements were used to characterize the novel QDs-labeled PBCN by morphology, particle size, size distribution, and zeta potential. The preliminary investigations on putative cytotoxic effect of QDs on cultured cells were performed using MTT assay. The suitability of QDs embedded in PBCN for fluorescent imaging of cells was also verified. The results obtained suggest that these hybrid nanoparticles have potentials for biological imaging applications. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Search for biomarkers to combat today’s most prevalent diseases strongly stimulates biomarker discovery and development in the last decade. Advances in nanotechnology have led to the development of semiconductor quantum dots (QDs), tiny lightemitting nanoparticles that emerge as a new class of fluorescent probes [1]. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties due to the so-called quantum confinement effects [2,3]. The first utilization of QDs as fluorescent biolabels in 1998 [4,5] has promoted further extensive research in this area for in vivo and in vitro biomolecular and cellular imaging. [6,7]. In contrast with organic fluorochromes, QDs imaging probes possess long-term photostability, an important feature that opens the possibility of investigating the dynamics of cellular process over time [8], such as continuously tracking cell differentiation [9] and metastasis [1]. The synthesis of QDs in organic

∗ Corresponding author. Tel.: +359 2 8161 387; fax: +359 2 962 5438. E-mail address: [email protected]fia.bg (C. Dushkin). 0927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2009.02.027

solvents at high temperature is one of the most mature and popular ways to achieve high-quality nanocrystals [10-15]. However, the as-prepared QDs are not hydrophilic and biocompatible. In order to exploit the unique properties of these nanoparticles in biosensing and biomedical imaging applications, they need hydrophilization and biocompatibilization. There have been reports on the surface modification of QDs, for example, conjugation of small molecules such as mercaptoacetic acid to QDs [5] and coating with silica on the QDs [16]. Other methods involve encapsulating QDs into micelles [9,17], liposomes [18,19] and amphiphilic polymers [20,21]. However, some of the above mentioned methods for transferring QDs into water have the additional disadvantages of being rather complicated and time-consuming, and the intrinsic instability of micelles and liposomes may lead to leaking of QDs into solution. Furthermore, some encapsulated QDs did not maintain their free solution optical properties, since the emission spectra broaden and the emission intensity severely quench. On the other hand, poly(alkylcyanoacrylate) (PACA) nanoparticles are considered as one of the most promising polymer carriers for drug delivery. These nanoparticles meet ideally the requirements for therapeutic and biomedical applications due to

200

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

a number of properties such as biocompatibility, biodegradability, low toxicity, easy preparation and purification, ability to alter drug biodistribution and to overcome some biological barriers [22–24]. It has previously been shown the ability of PACA nanoparticles to overcome the drug resistance in cancer cells [22], to facilitate the transport of drugs into the brain [23,24], and also to enhance skin penetration of drugs [25–27]. The mechanisms of PACA nanoparticles interactions with different biological cells depend on many factors and are in some cases still under discussion. Fluorescent tagging of PACA nanoparticles offers a possibility to gain an insight into these mechanisms by visual tracking of the labeled nanoparticles. In this study, highly fluorescent CdSe/CdS core-shell QDs were synthesized, and then embedded into poly(butylcyanoacrylate) nanoparticles (PBCN) in order to prepare novel fluorescent nanocomposite particles for bioimaging applications. The listed above properties and advantages of poly(alkylcyanoacrylates) make them appropriate materials for biocompatibilization of QDs for potential biological and diagnostic applications. The encapsulation of QDs is of benefit in solving some problems associated with QDs such as biocompatibility and stability. This could open a possibility to prepare a platform by incorporating together bioactive compounds and labeling agents that allow investigation of the mechanisms of carrier–cell interactions, penetration and localization of the polymer nanoparticles in various biological cells. 2. Experimental 2.1. Chemicals 2-Butylcyanoacrylate monomer was from Special Polymers Ltd. (Bulgaria). Dextran 40 was from Pharmachim (Bulgaria). Pluronic F68 (PEO-PPO-PEO triblock co-polymer) was from Sigma. Cadmium oxide (CdO, 99%), sulfur (S, purum, 99.5%), and pulverized selenium (Se, pure, 99.5%) were from Fluka. Tributylphosphine (TBP, 97%) was from Aldrich. Liquid paraffin and stearic acid were from RA.M.Oil SpA (Italy) and Hatkim SA (Turkey), respectively. Chloroform was of analytical reagent grade from Labscan Ltd. (Ireland). All other chemicals and solvents were used as received, without additional purification. Tributylphosphine sulfide (TBP-S) solution (0.125 M) and tributylphosphine selenide (TBP-Se) solution (0.125 M) in liquid paraffin were used as chalcogenide precursors for the synthesis of QDs. 2.2. Preparation of QD-loaded PBCN (QD/PBCN) The novel hybrid (QDs/PACA) nanoparticles, presented in this paper, were prepared by interfacial polymerization of butyl-2-cyanoacryale monomer in aqueous medium containing luminescent QDs. First, core/shell CdSe/CdS QDs were prepared in liquid paraffin and purified as described in ref. [28]. The obtained CdSe/CdS QDs were made water-dispersible by solubilization with the amphiphilic triblock copolymer Pluronic F68. For the successful solubilization, QDs (7 mg) were dissolved in chloroform (20 ml) solution of Pluronic F68 (500 mg) upon ultrasound sonication to form clear dispersion and then rotary evaporated. The residue in the flask was further dried under vacuum (∼10−3 atm) for 20 min. Distilled water (50 ml) was added and the obtained dispersion was centrifuged (6000 rpm, 10 min) to remove nondispersed aggregates. The final dispersion of QDs in the Pluronic F68 solution (QDs/F68) was visibly clear (without opalescence) and fluoresced upon UV-light (365 nm) illumination. Butyl-2cyanoacrylate monomer (50–100 ␮l) dissolved in dry acetone (2.5 ml) was added dropwise to the aqueous dispersion of QDs-F68 (5 ml) upon magnetic agitation (∼400 rpm) at room temperature. The polymer walls form immediately onto the QDs leading to the

hybrid nanoparticles (QDs/PBCN). The pH in was ∼7 in all preparations. The dispersions were stirred for 3 h at room temperature for complete polymerization. Then the acetone was evaporated. 2.3. Preparation of pure PBCN For the preparation of PBCN, the monomer butylcyanoacrylate was polymerized in aqueous polymerization medium containing Pluronic F68 (1%, wt./v) with and without presence of dextran 40 (4 mg/ml). For that purpose the butylcyanoacrylate monomer (50–100 ␮l) was dissolved in dry acetone (2.5 ml) and was added dropwise to water solution of Pluronic F68 (1 wt.%, 5 ml) upon magnetic stirring. The resulting milky dispersions were stirred for 3 h at room temperature for complete polymerization. 2.4. Methods for analysis The absorbance of QDs suspensions was measured by using a UV–vis spectrophotometer Jenway 6400. The fluorescence spectra were measured by using a fluorescence spectrophotometer Cary Eclipse (Varian) (excitation at 480 nm). Quartz cuvettes were utilized in both absorbance and emission measurements. The sizes and ␨-potentials of PBCN and QDs/PBCN were determined in 1 mM NaCl by electrophoretic light scattering system ZETASIZER IIC (Malvern Instruments, UK). The average particle sizes were calculated based on the average values of three or four measurements. Light scattering system for dynamic and static light scattering Malvern 4700C (Malvern Instruments, UK) was used to evaluate the nanoparticle size distributions. All light scattering experiments have been carried out on nanoparticle dispersions in water at 25 ◦ C. The images of transmission electron microscope (TEM) and scanning transmission electron microscope (STEM) were inspected by JEM-2100F (JEOL) operated at 200 kV of acceleration voltage. An energy dispersive X-ray spectroscopy (EDS) and element mapping were performed by the same TEM in STEM mode with the probe size of 1.5 nm. To study the photostability of water dispersions of QDs/F68 (absorbance at 580 nm was 0.08) and QDs/PBCN were illuminated with UV-light (maximum emission at 370 nm; the light power density at the sample position was 2.5 mW/cm2 ) for maximum 4 days at room temperature. The respective control samples were kept on ambient light. Fluorescence spectra of the UV-illuminated and control samples were measured. Light radiation was measured with Research Radiometer (Ealing Electro-optics, Inc.). 2.5. MTT assay The investigations on putative cytotoxic effect of QDs on cultured cells were performed using 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The Chinese hamster ovary (CHO) cell line was used in the experiments. The cells were seeded at a concentration of 4 × 104 cells/well in 96-well plates (Cellstar) in Ham’s F12 medium (Sigma), supplemented with 10% fetal bovine serum (Cambrex, Belgium), 100 U/ml penicillin and 100 ␮g/ml streptomycin, and cultured at 37 ◦ C in a humidified CO2 incubator. At the 24th h the cells from monolayers were washed and covered with media modified with different concentrations (each concentration in 4 repetitions) of QDs. Samples of cells grown in non-modified medium served as a control. MTT assay was performed as described by Mosmann [29] after 4, 24 and 48 h incubation periods. Optical density was measured at wavelength 540 nm by Absorbance Reader Tecan. Relative cell viability, expressed as a percentage of the untreated control, was calculated for each concentration used.

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

201

Statistical analysis: The data are presented as mean ± standard error of the mean (SEM). Statistical differences between control and treated groups were assessed using one-way analysis of variance (ANOVA) followed by Dunnett post-hoc test. 2.6. Fluorescent visualization of QDs/PBCN in cultured cells The suitability of the new hybrid nanoparticles for cell imaging was also verified. CHO cells were seeded on coverslips in 6-well plates (Cellstar) and cultured in the above-mentioned conditions. At the 24th h cells from monolayers were washed and covered with media modified with aqueous hybrid nanoparticles suspension, containing 10 mg/ml poly(butylcyanoacrylate) and 0.05 mg/ml QDs. After 3 h of incubation at 37 ◦ C in a humidified CO2 incubator the coverslips were fixed in methanol (3 min) and airdried. Then the cells were examined by fluorescent microscopy. The examination was performed in epi-illumination NU2 system using HBO200 mercury lamp for light source, excitation filter B224G and the combination of barrier filters G245 + G249. 3. Results and discussion Normalized absorbance and fluorescence spectra of core CdSe and the respective CdSe/CdS core/shell QDs to be embedded in PBCN are shown in Fig. 1. The growth of CdS shell leads to red shifts in the QDs spectra, evident for the successful preparation of core/shell structures. The core/shell CdSe/CdS QDs were also spheroids of average size 4.3 nm (with a CdSe cores of average size 3.1 nm) (Fig. 2a). TEM analysis confirmed the larger size of the final CdSe/CdS QDs in comparison with the initial CdSe cores. Also, TEM/EDS of CdSe/CdS QDs confirmed their elemental composition: cadmium, selenium and sulfur. The full width of the fluorescence band was 33 nm and the relative quantum yield (QY) was found to be 65%. During the transfer to water by means of Pluronic F68, single QDs as well as some aggregates (<40–50 nm in size) are formed as evident from the TEM studies (Fig. 2b). The hybrid nanoparticles (QDs/PBCN) are with spherical shape (Fig. 3). Aggregates of QDs embedded in PBCN are clearly seen by TEM. The electron dispersion spectroscopy (EDS) for cadmium from the QDs/PBCN samples was relatively low and allowed obtaining

Fig. 1. Normalized (a) absorbance and (b) fluorescence spectra of CdSe cores and CdSe/CdS core/shell QDs utilized in our experiments.

Fig. 2. TEM image of (a) CdSe/CdS QDs; (b) QD-F68. Size bars represent 10 nm.

of reliable mapping by scanning TEM (STEM) only for larger QDaggregates incorporated in PBCN (Fig. 4). QDs were found to be non-homogeneously distributed in the PBCN. It was very difficult to detect single QDs or very small aggregates of QDs embedded in PBCN, because of the low intensity of the EDS signal for cadmium mapping. For that reason it was not possible to determine the fractions of QDs-labeled and no labeled PBCN in a sample. There were QDs/PBCN, which contain larger (∼50 nm) or smaller aggregates of QDs, and PBCN, which contain single QDs. The encapsulation efficiency of QDs in the polymer could not be counted either by filtration or centrifugation. Our experience shows that the QDs coagulate upon contact with the filter membrane (100 nm pore size). Also, QDs and polymer nanoparticles could not be separated by centrifugation, which did not allow determination of the encapsulation efficiency. In order to encapsulate all QDs in polymer, we utilized relatively large excess of monomer (see Section 2). Studies by TEM confirmed that almost all QDs were successfully embedded inside the PBCN particles. Despite of the above difficulties, very clear images of QDs/PBCN were observed by fluorescent microscopy, which indicate that large enough number of PBCN have been successfully tagged with QDs in order to observe their interaction with biological cells. The aqueous dispersion of QDs/F68 is optically clear (Fig. 5a), whereas the dispersion of

202

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

Fig. 3. (a) TEM photograph of QDs/PBCN; size bar is 100 nm; (b) the respective size distribution determined by DLS.

QDs/PBCN is cloudy (Fig. 5b). Both dispersions were fluorescent upon UV-illumination. Investigation by TEM/EDS confirmed that the QDs were embedded inside the PBCN particles. We suppose that the driving force to form the hybrid nanoparticles or binding force between QDs and polymers is based on some kind of affinity between the QD-surface and the polymer. For example, the cyano-groups (–CN) from the polymer may coordinate to the Cd(II) species from the QD-surface. The formation of such coordination bonds could also explain the protective effect of the polymer against photo-bleaching and the photo-induced annealing of surface defects in QDs (see below). Various in size QDs/PBCN (100–200 nm) can be reproducibly prepared by varying the concentration of butylcyanoacrylate monomer (Fig. 6). As expected, with increasing of the monomer concentration the QDs/PBCN diameter increases as well. The size and size distribution of the nanoparticles were determined by dynamic light scattering (DLS), whereas their ␨-potential was measured by electrophoretic light scattering in 1 mM NaCl. No significant differences in nanoparticle size and ␨-potential between hybrid QDs/PBCN and PBCN were observed (Table 1). The ␨potential in all cases was found to be from −13 to −17 mV and does not depend on the nanoparticle size and the presence of dextran in the polymerization medium. In expectation of these findings the interaction of both QDs/PBCN and PBCN with living cells will be

Fig. 4. STEM of a single QDs/PBCN: (a) STEM image; (b) Cd mapping (L␣ line); (c) Se mapping (L␣ line).

analogous due to their similar size, size distribution and ␨-potential as well as the same nature of their contact surfaces. The obtained hybrid QDs/poly(butylcyanoacrylate) nanospheres are stable colloids for at least 1 month. Samples containing dextran are found to Table 1 Size and ␨-potential of PBCN and QD/PBCN, prepared with and without addition of dextrane. The monomer concentration is 15 ␮l/ml. Standard deviations from three or four measurements are given. Stabilizer

NPs

Size (nm)

␨-Potential (mV)

Pluronic F68

PBCN QD/PBCN

130.3 ± 4.9 128.1 ± 3.8

−17.54 ± 0.22 −13.22 ± 0.66

Pluronic F68 + dextrane

PBCN QD/PBCN

123.2 ± 3.3 134.3 ± 2.1

−16.05 ± 0.42 −16.24 ± 0.32

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

203

Fig. 7. Fluorescence spectra of (a) QDs/F68, and (b) QDs/PBCN (average size 180 nm) in water, before and after 48 h of UV-illumination (maximum emission at 370 nm; the light power density at the sample position was 2.5 mW/cm2 ). Photo-bleaching is observed for the case of QDs/F68. The fluorescence intensity from QDs/PBCN increases nearly twice during the UV-illumination due to a photo-activation phenomenon. The fluorescence images from the respective water dispersions are shown as insets.

Fig. 5. Photographs of water dispersions of nanoparticles at ordinary light (left) and upon UV-illumination (right): (a) QDs/F68; (b) QDs/PBCN.

be even more stable and do not form any aggregates for at least 3–4 months because of its colloid stabilizing role. In all cases, the fluorescence of QDs/PBCN remains unchanged for months when stored at room temperature, in ambient light

Fig. 6. Diameter of PBCN and QDs/PBCN as a function of butylcyanoacrylate (BCA) monomer per millilitre solution of Pluronic F68 (1 wt.%). The sizes are measured by photon correlation spectroscopy in 1 mM NaCl.

and open air. The photostability tests show that the coating of QDs with poly(butylcyanoacrylate) results in extremely good protection of QDs against photo-oxidation upon UV-illumination. QDs/F68 aqueous dispersions are stable on open air and ambient light, but photo-bleached upon UV-light (Fig. 7). In comparison, the fluorescence intensity of QDs/PBCN increases almost twice after 48 h of illumination and remains almost constant until the end (4 days) of experiment. The reason for the increase of fluorescence intensity in this case is probably the photo-induced annealing of the surface defects of QDs. Similar excitation dependent fluorescence has been previously observed from CdSe QDs in monolayers and solutions [30-34]. The annealing of the surface defects of QDs could be a result from formation of coordination bonds between the –CN groups of polymer and the Cd(II) species on the QD-surface. The resistance of QDs/PBCN to photo-bleaching could be considered as indication for a successful embedding of QDs in PBCN leading to protection against photo-oxidation. The investigations performed by MTT assay revealed that bare QDs exerted a time- and concentration-dependent effect on the viability of CHO cells (Fig. 8). The obtained preliminary results indicate that the viability of cells cultured in the presence of QDs applied at concentrations equal to or minor than 6.25 ␮g/ml for all treatment periods (4, 24 and 48 h) examined is equal to or bigger than 80%. The higher concentrations tested (12.5, 25 and 50 ␮g/ml) were found to be relatively non-toxic (cell viability ≥70%) only after a 4 h period (Fig. 8a). Further incubation of cells in the presence of QDs applied at these concentrations was accompanied by a strong and progressive cytotoxic activity (Fig. 8b and c). CHO cells, treated with both QDs and QDs/PBCN were studied by fluorescence microscopy to illustrate their suitability for cell imaging. Lack of ability of QDs for membrane cell accumulation was observed and visualized in Fig. 9a, which could be a possible reason for the low effect of QDs on cell viability after 4 h incubation. Conversely, bright granular orange–red fluorescence was observed on the surface of CHO cells treated with QDs/PBCN demonstrating their affinity to the cell surface (Fig. 9b).

204

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

Fig. 9. Fluorescence microscopy images of Chinese hamster ovary cells treated with QDs (a) and QDs/PBCN (b). Each tiny fluorescent spot is attributed to a single QDs/PBCN (b). No background fluorescence from the cells is detected.

4. Conclusions

Fig. 8. Time- and concentration-dependent effect of bare QDs on the viability of Chinese hamster ovary cells determined by MTT assay: Cells were incubated with QDs for (a) 4 h; (b) 24 h and 48 h (c). Data represent mean ± SEM (*P < 0.05; **P < 0.01).

The affinity of PBCN to the cell surface is an important feature when utilizing these nanoparticles in drug delivery, because it leads to larger effective concentration of drug close to the cell membrane. The affinity of the PBCN to the cell membrane could depend on various factors, such as nanoparticle size, ␨-potential, type of loaded drug and type of surface modification. Utilization of the novel hybrid QDs/PACA nanoparticles in future experiments is expected to reveal the effects of all these factors on the mechanisms of nanoparticle–cell interactions.

We developed the above-described new hybrid (QDs/PACA) nanoparticles for the needs of cell imaging. These formulations combine the important qualities of both QDs (excellent optic/fluorescent properties) and PACA (biocompatibility, low toxicity). The successful embedding of QDs in PACA nanoparticles during controlled anionic polymerization in aqueous media results in the formation of fluorescent hybrid (inorganic/organic) nanocomposite particles, which could be effectively utilized in biological imaging. The utilization of QDs as fluorescent labels for PACA nanoparticles is an advantageous strategy, which could allow an effective tracing of the interaction between the QDs-tagged PACA nanoparticles and biological cells. The accumulation of the hybrid (QDs/PACA) nanoparticles on cell membranes was clearly observable by fluorescent microscopy in the preliminary studies performed. We expect that this new imaging tool will permit the investigation and knowledge of some biological processes in various biological cells, the nanoparticle–cell interactions, the localization and mechanisms of action of drug-loaded nanoparticles in different cell lines. However, further investigations are needed to clarify completely the biological behavior, safety and possible applications of both QDs and QDs/PBCN. These new materials may reveal new phenomena and new properties, which are of both fundamental and practical importance.

G. Yordanov et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 339 (2009) 199–205

Acknowledgements The financial supports of the Bulgarian Ministry of Education and Science (Projects DO 02-168 and VUH-09/05) and the University of Sofia (Project 89/2008) are greatly acknowledged. Part of this work was supported by the “High-Tech Research Center” project for private universities; matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors are thankful to Dr. Marin Alexandrov (Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences) for the kind assistance with fluorescent microscopy images. References [1] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Quantum dots for live cells, in vivo imaging and diagnostics, Science 307 (5709) (2005) 538–544. [2] A.D. Yoffe, Semiconductor quantum dots and related systems: electronic, optical luminescence and related properties of low dimensional systems, Adv. Phys. 42 (2) (1993) 173–266. [3] S.V. Gaponenko, Optical Properties of Semiconductor Nanocrystals, Cambridge University Press, 2005. [4] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Semiconductor nanocrystals as fluorescent biological labels, Science 281 (1998) 2013–2016. [5] W.W. Chan, S. Nie, Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science 281 (1998) 2016–2018. [6] F. Pinaud, X. Michalet, L.A. Bentolila, J.M. Tsay, S. Doosel, J.J. Li, G. Iyer, S. Weiss, Advances in fluorescence imaging with quantum dot bio-probes, Biomaterials 27 (2006) 1679–1687. [7] T. Jamieson, R. Bakhshi, D. Petrova, R. Pocock, M. Imani, A.M. Seifalian, Biological applications of quantum dots, Biomaterials 28 (2007) 4717–4732. [8] M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, A. Triller, Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking, Science 302 (2003) 442–445. [9] B. Dubertret, P. Skourides, D.J. Norris, V. Noireaux, A.H. Brivanlou, A. Libchaber, In vivo imaging of quantum dots encapsulated in phospholipid micelles, Science 298 (2002) 1759–1762. [10] C.B. Murray, D.J. Norris, M.G. Bawendi, Synthesis and characterization of nearly monodisperse CdE (E = S, Se Te) semiconductor nanocrystallites, J. Am. Chem. Soc. 115 (1993) 8706. [11] Z.A. Peng, X. Peng, Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor, J. Am. Chem. Soc. 123 (2001) 183–184. [12] L. Qu, Z.A. Peng, X. Peng, Alternative routes toward high quality CdSe nanocrystals, Nano Lett. 1 (6) (2001) 333–337. [13] J. Jasieniak, C. Bullen, J. van Embden, P. Mulvaney, Phosphine-free synthesis of CdSe nanocrystals, J. Phys. Chem. B 109 (2005) 20665–20668. [14] W.W. Yu, X. Peng, Formation of high-quality CdS and other II-VI semiconductor nanocrystals in noncoordinating solvents: tunable reactivity of monomers, Angew. Chem. Int. Ed. 41 (13) (2002) 2368. [15] G. Yordanov, H. Yoshimura, C. Dushkin, Phosphine-free synthesis of metal chalcogenide quantum dots by means of in situ-generated hydrogen chalcogenides, Colloid Polym. Sci. 286 (2008) 813–817.

205

[16] P. Mulvaney, L.M. Liz-Marzan, M. Giersig, T. Ung, Silica encapsulation of quantum dots and metal clusters, J. Mater. Chem. 10 (2000) 1259–1270. [17] H. Li, X. Wang, Z. Gao, Z. He, Gemini surfactant for fluorescent and stable quantum dots in aqueous solution, Nanotechnology 18 (2007) 205603. [18] L. Feng, X. Kong, K. Chao, Y. Sun, Q. Zeng, Y. Zhang, Efficient phase transfer of hydrophobic CdSe quantum dots: from nonpolar organic solvent to biocompatible water buffer, Mater. Chem. Phys. 93 (2005) 310–313. [19] C. Chen, J. Yao, R. Durst, Liposome encapsulation of fluorescent nanoparticles: Quantum dots and silica nanoparticles, J. Nanopart. Res. 8 (6) (2006) 1033–1038. [20] T. Nann, Phase-transfer of CdSe@ZnS quantum dots using amphiphilic hyperbranched polyethylenimine, Chem. Commun. (2005) 1735–1736. [21] A.F.E. Hezinger, J. Teßmar, A. Gopferich, Polymer coating of quantum dots—a powerful tool toward diagnostics and sensorics, Eur. J. Pharm. Biopharm. 68 (2008) 138–152. [22] R.S.R. Murthy, L.H. Reddy, Poly(alkyl cyanoacrylate) nanoparticles for delivery of anti-cancer drugs, in: M.M. Amiji (Ed.), Nanotechnology for Cancer Therapy, CRC Press, 2006, pp. 251–288. [23] K. Ringe, C.M. Walz, B.A. Sabel, Nanoparticle drug delivery to the brain, in: H.S. Nalwa (Ed.), Encyclopedia of Nanoscience and Nanotechnology, 7, American Scientific Publishers, 2004, pp. 91–104. [24] D.J. Begley, M.W. Bradbury, J. Kreuter (Eds.), The Blood–Brain Barrier and Drug Delivery to the CNS, Marcel Dekker Inc., New York, 2000. [25] S. Miyazaki, A. Takahashi, W. Kubo, J. Bachynsky, R. Löbenberg, Poly nbutylcyanoacrylate (PNBCA) nanocapsules as a carrier for NSAIDs: in vitro release and in vivo skin penetration, J. Pharm. Pharmaceut. Sci. 6 (2) (2003) 238–245. [26] M. Simeonova, R. Velichkova, G. Ivanova, V. Enchev, I. Abrahams, Poly(butylcyanoacrylate) nanoparticles for topical delivery of 5-fluorouracil, Int. J. Pharm. 263 (1-2) (2003) 133–140. [27] M. Hadjikirova, P. Troyanova, M. Simeonova, Nanoparticles as drug carrier system of 5-fluorouracil in local treatment of patients with superficial basal cell carcinoma, J. B. U. O. N. 10 (4) (2005) 517–521. [28] G. Yordanov, H. Yoshimura, C. Dushkin, Synthesis of high-quality core/shell quantum dots of CdSe/CdS by means of gradual heating in liquid paraffin, Colloid Polym. Sci. 286 (2008) 1097–1102. [29] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay, J. Immunol. Methods 65 (1983) 55–59. [30] S. Maenosono, C. Dushkin, S. Saita, Y. Yamagushi, Optical memory media based on excitation-time dependent luminescence from a thin film of semiconductor nanocrystals, Jpn. J. Appl. Phys. 39 (2000) 4006–4012. [31] S.R. Cordero, P.J. Carson, R.A. Estabrook, G.F. Strouse, S.K. Buratto, Photoactivated luminescence of CdSe quantum dot monolayers, J. Phys. Chem. B 104 (2000) 12137–12142. [32] Y. Wang, Z. Tang, M.A. Correa-Duarte, I. Pastoriza-Santos, M. Giersig, N.A. Kotov, L.M. Liz-Marzan, Mechanism of strong luminescence photoactivation of citratestabilized water-soluble nanoparticles with CdSe cores, J. Phys. Chem. B 108 (2004) 15461–15469. [33] M. Jones, J. Nedeljkovic, R.J. Ellingson, A.J. Nozik, G. Rumbles, Photoenhancement of luminescence in colloidal CdSe quantum dot solutions, J. Phys. Chem. B 107 (2003) 11346–11352. [34] G. Yordanov, G. Gicheva, C. Dushkin, Optical memory based on photo-activated fluorescence of core/shell CdSe/CdS quantum dots embedded in poly(butylmethacrylate), Mater. Chem. Phys. 113 (2009) 507–510.