Highly fluorescent ethyl cellulose nanoparticles containing embedded semiconductor nanocrystals

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Highly fluorescent ethyl cellulose nanoparticles containing embedded semiconductor nanocrystals Alla N. Generalova a,∗ , Svetlana V. Sizova a , Vladimir A. Oleinikov a , Vitaly P. Zubov a , Michail V. Artemyev b , Liat Spernath c , Alexander Kamyshny c , Shlomo Magdassi c a b c

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, Moscow 117871, Russia Belurussian Institute for Physico-Chemical, Problems, Leningradskaya Str. 14, Minsk 220080 Belarus Casali Institute of Applied Chemistry, Institute of Chemistry, The Hebrew University of Jerusalem, Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel

a r t i c l e

i n f o

Article history: Received 18 December 2008 Received in revised form 1 April 2009 Accepted 2 April 2009 Available online 10 April 2009 Keywords: Nanoemulsions Ethyl cellulose nanoparticles EIP method Semiconductor nanocrystals Quantum dots Fluorescent nanoparticles Particle agglutination

a b s t r a c t Highly luminescent organic nanoparticles were formed by embedding hydrophobic and hydrophilic (CdSe)ZnS quantum dots with core/shell structure into ethyl cellulose nanoparticles. The nanoparticles were prepared from oil-in-water nanoemulsions by a phase inversion process at constant temperature, followed by a solvent evaporation. The obtained fluorescent ethyl cellulose nanoparticles were functionalized by immobilization of a specific antibody, and applied in rapid agglutination test for detection of Yersinia pestis F1-antigen. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Nanoparticles are of a great interest in a variety of scientific fields such as cell biology, biotechnology, diagnostics, analytics, and pharmaceutics [1–3]. In medicine, for example, functionalized nanoparticles find applications in sensing and diagnostics on a single-cell level [4]. Recent progress in polymer science allows preparation of mechanically stable, size- and shape-persistent polymer nanoparticles. The development of methods for producing fluorescent nanoparticles can open new fields for their application. Traditional fluorescent latex particles with organic dyes suffer from photobleaching and technical difficulties in multicolor analysis. Development of nanotechnological methods has made possible preparation of luminescent semiconductor nanocrystals, which are very promising alternative to organic dyes due to their excellent photostability, high quantum yield, and a narrow emission peak, the position of which depends only on the nanocrystal size (in the range of 2–8 nm) regardless of the excitation wavelength [5]. An example of such particles are (CdSe)ZnS nanocrystals containing the CdSe

∗ Corresponding author. Tel.: +7 495 3360600; fax: +7 495 3351011. E-mail address: [email protected] (A.N. Generalova). 0927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2009.04.007

central core, which determines the fluorescence parameters, and a ZnS shell, which provides a high quantum yield of the fluorescence [6]. Semiconductor nanocrystals, or quantum dots (QD), are perspective for multicolor labeling and simultaneous identification of various biological objects [7]. They emit in a wide spectral range, from blue to red, depending on their size that allows tuning the fluorescence color with the use of one excitation source. Among the polymers suitable for encapsulation of semiconductor nanocrystals, ethyl cellulose (EC) is very attractive. This hydrophobic polymer is non-toxic, stable, and has been widely used in pharmaceutics. This cellulose ether is a substantially waterinsoluble polymer, and is excessively used in coatings of solid dosage forms to provide a controlled release profile of drug substances [8,9]. This polymer is used in many other drug delivery systems, such as matrices [10], microspheres [11], microcapsules [12], also in combination with other cellulose derivatives [13–15]. EC being primarily applied in organic solvents is now also available as 30% colloidal polymer particles of 200–500 nm in diameter, dispersed in an aqueous phase [16]. EC latexes can be cast or sprayed onto the surface of the desired dosage forms, the dispersion is exposed to gradual water evaporation and polymer deformation. Upon more complete evaporation of the aqueous phase or the organic solvent, the polymer chains are aligned to further coalescence to form a homogeneous, transparent film [16].

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Attractive colloidal systems are nanoemulsions with droplet size in the nanometric scale (typically in the range 20–200 nm), which are stable against flocculation and sedimentation. The droplets of the nanoemulsions can be used as “nanoreactors” for chemical reactions [17] or as dissolving medium for preformed polymers [18], drug delivery system [19], and cosmetics [20]. Nanoparticles containing diagnostic tests (qualitative, yes/no results) and assays (quantitative results) are usually based upon the specific interaction of antigen (Ag) with antibody (Ab). Sub-micron sized nanoparticles are used as the solid support for immobilization of Ab or Ag onto them. These “sensitized” nanoparticles then act to magnify or amplify the Ag–Ab reaction that takes place when they are mixed with a sample containing an opposite reactant. In addition, the use of fluorescent-labeled nanoparticles allows increasing the analysis sensitivity and specificity [21]. When antigens are present in the analyte, the interactions of antigens with the immobilized antibodies result in the aggregation of nanoparticles. Aggregation can be observed with the naked eye or monitored quantitatively by any physicochemical method sensitive to the size of aggregates. In simple particle agglutination test, a positive test results when uniformly dispersed milky-appearing Ab-coated particles in a drop of buffer on a glass slide react with Ag in a drop of sample (body fluids) to cause particle agglutination (clumping of the nanoparticles to look like curdled milk). These so called “glass slide tests” are portable, rapid, efficient, and useful even under the most poorly controlled conditions. Ideal for point-of-care use in the field, ambulance, or bedside, they can be performed quickly and simply (5 min from sample preparation) [2]. In this work we present a new process for preparation of EC nanoparticles containing embedded fluorescent probes: two types of (CdSe)ZnS QD with size of 3.5 and 6 nm emitting green and red light, respectively. Semiconductor nanocrystals embedded into EC particles can potentially be used not only as fluorescent markers, but additionally as luminescence sensors and ion probes. The EC nanoparticles were produced by a solvent evaporation method from oil-in-water nanoemulsion prepared by phase inversion at constant temperature [22]. The possibility to use the fluorescent EC particles conjugated with antibody as a bioanalytical reagent in the particle agglutination test was also evaluated.

2. Material and methods The following materials were purchased from Sigma and used without further purification: ethyl cellulose with a viscosity of 45 cPs for a 5% solution in 4:1 toluene:ethanol at 25 ◦ C, sorbitan monolaurate (Span 20), mercaptoacetic acid, mercaptoethanol, Nhydroxysuccinimide, 1-ethyl-3-(3-dimetylaminopropyl) carbodiimide hydrochloride, Na-borate buffer, NaCl. Decaglycerol monolaurate was obtained by the courtesy of Sakamoto Yakuhin Kogyo Co. Ltd., Japan. Toluene, ethanol, methanol, dimethyl sulphoxide, chloroform were of analytical grade and purchased from Aldrich. Semiconductor nanocrystals CdSe/ZnS with core/shell structure (CdSe central core which determines parameters of fluorescence and ZnS-shell which provides a high quantum yield of fluorescence and prevents quenching) stabilized with tri-n-octylphosphine oxide (TOPO) were synthesized as described previously [23]. In the present work hydrophobic nanocrystals of diameters of 3.5 nm with emission peak 546 nm and of 6.0 nm with emission peak 620 nm were used and excited with ex = 380 nm. Anti-Y. pestis monoclonal mouse antibody (clone F-19) and Y. pestis F1-antigen of the plague bacterium, Yersinia pestis which is fibrillar protein collapsed into an antiphagocytic capsule-like structure on the surface of the bacterium were gratefully donated by Prof. Sveshnikov P.G., RCMDT, Moscow.

The size of particles was measured by photon correlation spectroscopy (Coulter N4-MD), optical and fluorescent characteristics were measured with the use of Zeiss Axiovert 200 microscope, Shimadzu RF-551 spectrofluorimeter, BioDoc-IT System UV-Transilluminator. High resolution SEM measurements were performed with the use of Sirion electron microscope (FEI Company). 2.1. Preparation of EC nanoparticles The EIP method is based on dropwise addition of water to a mixture of oil and surfactants while stirring that results in formation of o/w nanoemulsion with small and uniform droplet size [22]. More specifically, the surfactants (decaglycerol monolaurate and Span 20) were added to a 10% (w/w) solution of ethyl cellulose in toluene. Then the aqueous phase (10 mM NaCl) was dropwise added, at a constant rate of 0.5 ml/min. The addition of the aqueous phase was performed under continuous stirring at room temperature. The final nanoemulsion contained 20% (w/w) toluene and 5% (w/w) surfactant mixture. After the nanoemulsion was formed, the toluene was evaporated under reduced pressure (1 mmHg) at 45 ◦ C for 30 min, resulting in the formation of ethyl cellulose nanoparticles. The samples before and after evaporation were analyzed by gas chromatograph for the presence of toluene. Toluene was extracted from the samples using dichloromethane prior to analysis. GC analysis was performed using a HP 5890 gas chromatograph with a 5% diphenyl, 95% dimethylpolysiloxane column (30/0.25 m). 2.2. Swelling of EC nanoparticles in water–ethanol medium The study of EC particles swelling allows optimization of the procedure of QD embedding. Selection of the water–ethanol mixture was based on its ability to effectively disperse the fluorescent nanocrystals and to “loosen” EC nanoparticles to make easier the nanocrystal penetration into particles while preserving their colloidal stability. Swelling of EC nanoparticles was carried out in glass cuvette by dropwise addition of ethanol to the dispersion of EC nanoparticles in water, stirring and incubation as described below. The swelling experiments were performed as follows. Aqueous dispersion of EC nanoparticles (200 ␮l, 2% w/w) was diluted with water (2 ml). Then, to this sample (sample 1), ethanol was added (three times in 0.05 ml) and after each addition, the dispersion was incubated for 30 min (sample 2), or 0.15 ml of ethanol was added at once (sample 3). The total incubation time for samples 2 and 3 was 90 min. 2.3. Embedding of hydrophobic nanocrystals into EC nanoparticles Ethanol (100 ␮l) was added dropwise to EC dispersion (1 ml). QDs (0.06 mg) with diameter of 3.5 nm and emission maximum at 546 nm stabilized with TOPO were purified by dispersing in chloroform and precipitating with methanol. Purified QDs were dispersed in ethanol (10 ␮l), and the fluorescence intensity was measured. Then the dispersion of QDs was added to 200 ␮l of EC nanoparticles dispersion in water–ethanol, and the mixture was stirred vigorously, sonicated for 5 min and incubated for 20 min while stirring (this procedure was repeated three times), shaken for 2 h at room temperature and centrifuged at 10,000 rpm for 10 min (to remove free QDs). To remove ethanol, the obtained EC nanoparticles containing embedded QDs (EC-QDs) were dialyzed against water. The fluorescence of the resulting dispersion containing ECQDs was measured, and the obtained intensity was corrected taking into account the dilution during the embedding procedure.

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2.4. Preparation of water-dispersed hydrophilic nanocrystals

Table 1 Sizes of swollen EC particles (samples 1, 2, and 3).

Nanocrystals were purified from TOPO by addition of three-fold volume of chloroform followed by precipitation in methanol. The purified nanocrystals (5 mg) were dispersed in 2 ml chloroform. Thiol reagents (mercaptoacetic acid, 5 mg, and mercaptoethanol, 5 mg) in 0.2 ml H2 O were added to 0.2 ml methanol. Then the solution of thiol reagents was added dropwise to the dispersion of nanocrystals in chloroform, and two immiscible phases (water–methanol solution of thiol reagents at the top and nanocrystals in chloroform at the bottom) were thoroughly shaken. After nanocrystals were transferred from one phase to another, chloroform was evaporated. To remove an excess of thiol reagents, the obtained nanocrystals suspended in water were washed with methanol and then three times with water (consecutive centrifugation–redispersion steps). Purified nanocrystals were dried in vacuum at room temperature and redispersed before using.

Sample 200 ␮l EC in 2 ml H2 O

Incubation time (min)

Added ethanol (␮l)

Mean diameter (nm)

1

–

–

112 ± 4.5

2

+50 +50 +50

30 30 30

132 ± 6.6 140 ± 8.2 152 ± 9.7

3

+150

30 90

148 ± 10.4 148 ± 15 77 ± 5.1

Table 2 Sizes of EC particles after ethanol treatment and embedding the semiconductor nanocrystals. Sample

Unimodal analysis Mean diameter (nm)

2.5. Embedding of water-dispersed QDs into EC nanoparticles Aqueous dispersion of EC nanoparticles (200 ␮l) was mixed with ethanol (20 ␮l). 10 ␮l of aqueous dispersed of QDs (3 mg in 500 ␮l of water) was added to dispersion of EC in ethanol–water, and the mixture was sonicated for 20 min, stirred for 2 h at room temperature, sonicated again for 10 min and kept overnight at vigorous stirring at 4 ◦ C. Then obtained dispersion was centrifuged at 12,000 rpm for 10 min to remove free QDs (supernatant fraction), the precipitate was dried under vacuum and the resultant EC nanoparticles with incorporated QDs were redispersed in water (400 ␮l) under ultrasonic treatment. The fluorescence intensity of QDs was measured before their embedding within the EC particles (aqueous dispersion of QDs), and after the embedding procedure (dispersion of EC particles containing embedded QDs and supernatant fraction after centrifugation). The measured fluorescence intensity was corrected taking into account the changes in concentration during the embedding procedure. 2.6. Antibody immobilization on EC nanoparticles In order to bind the monoclonal antibody (F19) to Y. pestis F1-antigen to EC particles, active esters of EC were prepared by using 1-ethyl-3-(3-dimetylaminopropyl) carbodiimide hydrochloride activated with N-hydroxysuccinimide [24]. More specifically, 0.04 mM N-hydroxysuccinimide in 50 ␮l dimethyl sulphoxide was added to 100 ␮l of 5% dispersion of EC particles containing embedded semiconductor nanocrystals in 0.1 M Na-borate buffer (BB), pH 8.2, stirred for 30 min, and then 0.04 mM 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride in 50 ␮l H2 O was added followed by stirring for 1 h. At the next stage the mixture was centrifuged, the sediment of particles was redispersed in 200 ␮l of 0.1 M BB, and 1 ␮l of antibody solution (6 mg/ml) was added. The resulting mixture was vortexed and kept overnight at 4 ◦ C while stirring. To block the unreacted groups, 0.5 ml of ovalbumin solution in BB (10 mg/ml) was added. Then the reaction mixture was washed by centrifugation–redispersion three times to remove an antibody excess and redispersed in 0.5 ml of ovalbumin solution in BB (10 mg/ml). Dispersion of particles with concentration of 1% was used for further experiments. 2.7. Nanoparticles agglutination Agglutination reaction on glass was carried out by serial twofold dilution of Y. pestis F1-antigen in each drop of 0.1 M BB (pH 8.2). First drop contained 25 ␮l of buffered Y. pestis F1-antigen solution (40 ␮g/ml), the last – 25 ␮l of buffered Y. pestis F1-antigen solution (2.5 ␮g/ml) and one drop contained only buffer as a control. Then

EC EC after ethanol treatment EC-QDs, max em 546 nm EC-QDs, max em 610 nm

117 120 128 132

± ± ± ±

5.9 7.2 7.7 7.9

SD (nm) 42 39 47 38

25 ␮l of sensitized dispersion of EC particles (1%) was added to all drops, the glass was shaken and left at room temperature. Measurements were performed after 5 min under UV-light. Positive test appears like curdled milk with dot in the centre, and negative test appears as a homogeneous suspension. 3. Results and discussion 3.1. Swelling of EC nanoparticles Swelling of EC nanoparticles is a process, which is accompanied by increase of diameter due to solvent penetration into particles. While studying the swelling, ethanol was taken in excess as compared with its amount used for the embedding procedure. The effect of swelling on the size of EC particles is presented in Table 1. It was found that the particle diameter increased by more than 30% after water–ethanol mixture treatment. These data are in good agreement with data for polymer particle swelling reported in literature [21]. In addition, as follows from Table 1, the most effective swelling was observed at stepwise addition of the solvent (sample 2), while in the case of sample 3 at simultaneous addition of the solvent, the formation of second set of particles with diameter ∼77 nm was observed. 3.2. EC particles containing hydrophobic semiconductor nanocrystals Hydrophobic (CdSe)ZnS nanocrystals (3.5 nm, max em = 546 nm) were embedded into EC particles from suspension in ethanol after purification from TOPO. The diameters of the original EC particles, EC particles after treatment with ethanol and EC particles containing embedded hydrophobic QDs with an average diameter of 3.5 and 6.0 nm are presented in Table 2. The data reveals that the embedding procedure does not result in a noticeable increase in the EC particles size as compared with the swelling experiments, in which much higher concentrations of ethanol were used (Table 1). Effective embedding of QDs into ethyl EC particles is proved by the fact that there were no free QDs in the dispersion media after centrifugation, and the fluorescence intensity of the supernatant fraction after centrifugation and a solution after dialysis was vanishingly low. It is worth noting that the obtained EC-QDs particles

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Fig. 1. Fluorescence image (a) and optical image (b) of EC containing embedded hydrophobic nanocrystals (Zeiss Axiovert 200) 540 nm.

display green fluorescence (Fig. 1), the intensity of which was twice higher compared to the fluorescence intensity of free QDs under the same conditions before embedding into EC particles (Fig. 2). This fluorescence intensity increase can be explained by recovering the nanocrystal shell ZnS after QDs embedding into EC nanoparticles. It is known that ZnS shell can be damaged after solvent (e.g. chloroform) treatment that results in quenching of fluorescence, while the polymeric shell around the nanocrystals seems to repair the ZnS shell after embedding thus enhancing the fluorescence intensity [25]. It was also found that the emission maximum of QDs embedded into EC particles is blue shifted (6 nm) compared to the emission maximum for free QDs. The embedding of QDs into EC particles probably eliminates the QDs surface state emission that imparts the photochemical stability. It should be noted that the surface state emission is caused by mismatch of emission and absorption peaks (Stokes’s shift) of free nanocrystals, in our case 546 and 525 nm, respectively [26]. Therefore, elimination of surface state emission due to formation of the polymer shell results in approaching emission peak (540 nm of embedding nanocrystals) to absorption peak (525 nm) and blue shift (6 nm) was observed. The effect of photo-activation of semiconductor nanocrystals resulting in the enhancement of luminescence intensity and in blue-shifting of emission wavelength is a phenomenon reported in recent literature where QDs were modified with amphiphylic polymers [25], embedded into acrolein–styrene polymer particles [27], polymer films [28], silica [29], etc. The exact mechanism is not clear yet, but the authors have proposed mechanism including surface reconstruction of the surface shell of nanocrystals because any traps for photogenerated electron and hole should be avoided.

Fig. 2. Fluorescence intensity of the hydrophobic QDs with em = 546 nm in water–ethanol media (1) and EC containing embedded hydrophobic QDs (2).

Possible traps in QDs are, as a rule, surface atoms which must be optimally constructed and passivated with some polymers to get rid of traps. It seems that this mechanism can be applied to explain effects in EC particles containing QDs. (CdSe)ZnS nanocrystals with d ≈ 6 nm and max em = 605 nm were also embedded within the EC nanoparticles. The obtained EC particles with red fluorescence were characterized by a slight increase in mean diameter (Table 2) compared to the original EC particles and also by the increase of QDs fluorescence intensity (∼3 times) compared to the free nanocrystals (Fig. 3). High resolution SEM images of these nanoparticles are shown in Fig. 4 (left). As follows from this figure, the size distribution is rather wide, and nanoparticles with diameters of about 50–120 nm are clearly observed. 3.3. EC nanoparticles containing embedded water-dispersed semiconductor nanocrystals The hydrophilic semiconductor nanocrystals (3.5 nm, max em = 546 nm) were obtained by a ligand exchange reaction: replacement of TOPO for mercaptoacetic acid with mercaptoethanol. The hydrophilic QDs dispersed in water–ethanol (20:1) mixture were embedded into ethyl cellulose particles swollen in ethanol. This yielded highly fluorescent, stable dispersion of EC nanoparticles with an average diameter of 138 nm, which is practically the same as for hydrophobic nanocrystals. As follows from Fig. 4 (right), the size distribution for EC particles containing embedded hydrophilic semiconductor nanocrystals is also wide,

Fig. 3. Fluorescence intensity of the hydrophobic QDs with em = 605 nm in water–ethanol media (1) and EC containing embedded hydrophobic QDs (2).

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Fig. 4. HR-SEM images of EC nanoparticles containing embedded hydrophobic QDs with emission maxima at 605 nm (left) and hydrophilic QDs with emission maxima at 546 nm (right).

and nanoparticles with diameters of about 70–180 nm are seen in the image. The supernatant fraction after centrifugation did not contain free QDs (no detectable fluorescence in the supernatant fraction after centrifugation), indicating their complete embedding of hydrophilic nanocrystals into the EC particles. However, in the case of embedded hydrophilic QDs, the fluorescent intensity did not exceed the intensity of the native nanocrystals and was even slightly lower (Fig. 5). Probably, the partially destroyed ZnS shell is not recovered after nanocrystal embedding because of the hydrophilic nature of nanocrystals hinders the formation polymeric shell around the nanocrystals [28]. In conclusion, it appears that both approaches yield highly fluorescent EC nanoparticles containing embedded QDs (Fig. 6), the first one enables significant increase in the intensity of the fluorescence emission. 3.4. Bioanalytical application The obtained fluorescent EC nanoparticles can be used both as carriers for biological ligands and as markers in agglutination test of “Ag–Ab”. EC particles containing embedded hydrophobic QDs with diameter of 3.5 nm and max em = 546 nm were functionalized by immobilization of antibody to the Y. pestis F1-antigen onto their surface. As described in Section 2.6, immobilization was carried out through activated ester obtained by using N-hydroxysuccinimide and by further covalent coupling with Ab in the presence of carbodiimide derivative. The agglutination test of these Ab-coated particles with the pathogen was carried out on a glass slide as

Fig. 6. Samples of EC containing embedded hydrophobic (1) and hydrophilic (2) QDs illuminated by UV-light (ex = 365 nm).

described in Section 2.7. The test results observed after 5 min under UV-light. The positive test (flocculated particles) looks like curdled milk with a fluorescent dot, while the negative test appears like a homogeneous fluorescent suspension. Weak flocculation during agglutination reaction was observed at 5 ␮g/ml, so minimal detected concentration in this assay was 10 ␮g/ml Y. pestis F1antigen. 4. Conclusions A simple method for preparation of highly fluorescent, colloidally stable EC particles by embedding semiconductor nanocrystals was developed. By utilizing this method, a wide range of nanocrystals with fluorescence color depending only on their size makes it possible to obtain great variety of functionalized luminescent polymeric particles required for performing various assays. It was found that the embedding of hydrophobic nanocrystals yields EC particles with fluorescence intensity twice higher than that of the native nanocrystal. The application of the fluorescent EC particles was demonstrated in an agglutination test, in which antibodies were attached to the particle surface. We expect that this method can be further utilized in various diagnostic tests. Acknowledgements

Fig. 5. Fluorescence intensity of the hydrophilic QDs with em = 546 nm in water–ethanol media (1) and EC containing embedded hydrophilic QDs (2).

The work was financially supported by the EU Sixth frame Work Program (“Novel and Improved Nanomaterials, Chemistries and Apparatus for Nano-Biotechnology” NACBO, project 500804-2), by the Russian Foundation for Basic Research (Projects 07-04-

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