Compact PEGylated polymer-caged quantum dots with improved stability

Colloids and Surfaces A: Physicochem. Eng. Aspects 402 (2012) 72–79

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Compact PEGylated polymer-caged quantum dots with improved stability Pengfei Zhang a,b,c , Huanxing Han a,∗ a Department of Laboratory Diagnosis, and Translational Medical Research Center, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China b Zhejiang California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang 310029, China c Shanghai Allist Pharmaceuticals Inc., Zhangjiang, Shanghai 201203, China

a r t i c l e

i n f o

Article history: Received 16 December 2011 Received in revised form 9 March 2012 Accepted 13 March 2012 Available online 21 March 2012 Keywords: Quantum dots PEGylation Glutathione Luminescence Stability

a b s t r a c t Compact PEGylated polymer-caged quantum dots were prepared by covalently cross-linking the surface ligands using bifunctional poly(ethylene glycol). CdSe/ZnSe/ZnS quantum dots prepared in organic solvent via conventional synthesis method were firstly transferred to water phase by surface ligands exchange with glutathione molecules, and then cross-linked by bifunctional PEG through the carboxylamine coupling by carbodiimide method. Compared with small molecules capped QDs, polymer-caged QDs using PEG crosslinker have shown improved colloidal and pH stability, lower cell toxicity and nonspecific binding, and still maintain compact hydrodynamic diameters (<15 nm) and monodispersity. This surface crosslinking strategy can also be used to improve the stability of other similar nanoparticles. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Recently, quantum dots (QDs) as a new class of fluorophores have attracted considerable interest in the biological area for their unique optical properties, including high quantum yields (QYs), large molar extinction coefficients, exceptional photostability, tunable emission wavelength and broad absorption cross section [1–4]. Current QDs synthesis process evolved in the surfactant-mediated nucleation and growth, and the resultant QDs were coated with a layer of surfactant to protect them from further growth and the external environment. However, most surfactants also rendered QDs hydrophobic and prevent them from further biological functionalization [5,6]. The key barrier toward widespread application of QDs in biological environment has been the presence of a trade-off among desirable properties, including small size, high fluorescence and colloidal stability, high QYs (resistant to illumination and time), facile surface decoration and low non-specific binding [7,8]. There are several strategies reported to transfer hydrophobic QDs from organic soluble to water soluble by stabilizing with silica [9–12], small molecular ligands [2,13–15], lipids [16] or amphiphilic polymers [17,18]. The overall size of QDs mainly depends on the thickness of coating layer. Among those methods, multilayers of polymer or silica coated on surfactant capped QDs,

∗ Corresponding author. Tel.: +86 21 81886078, fax: +86 21 33110236. E-mail address: [email protected] (H. Han). 0927-7757/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2012.03.022

giving rise to an overall large particle size. The large size of nanoparticles can limit the diffusion of QDs in the cellular cytoplasm [19], as well as potentially alter the native behavior of labeled receptors. Smaller QDs can be approached by replacing the original surfactants with thio-bearing small molecules, such as thioacetic acid [20] and cystein [13], but the resulting QDs are suffering from instability due to the weak interaction of monothiols with the QDs surface [7]. Precipitation of QDs would occur with the dissociation of ligands from the surface of QDs. Some improvement on the colloidal stability has been made by using either dithiol ligands such as dihydrolipoic acid [14], or multidentate thiol ligands such as artificial peptides with multidentate cysteine residues [21], to achieve more tight binding to the surface of QDs [22,23]. In recent researches, QDs ligands exchanged with small molecules containing thio-based ligands, such as mercaptoundecanoic acid [24] and glutathione (GSH) [25], were crosslinked by lysine or glutathione via carboxyl-amine reaction using carbodiimide method. Compared with non-covalent coating, this covalently crosslinking polymer shells capped QDs have demonstrated better biocompatibility and environmental stability [24–26]. However, QDs coated with peptide have specific or nonspecific binding to cells [27], which are not desired properties for labeling and biological assays. While previous studies have shown that PEGylated QDs can reduce nonspecific binding for both in vitro and in vivo applications [28,29], and improve colloidal stability under various experimental conditions [26]. In this paper, a strategy for preparation of compact PEGylated polymer-caged QDs was reported. The polymer-caged QDs were achieved by covalently crosslinking of GHS capped QDs with

P. Zhang, H. Han / Colloids and Surfaces A: Physicochem. Eng. Aspects 402 (2012) 72–79

bifunctional PEG, which can be served as both crosslinkers and PEGylation agents. The crosslinking densities and PEGylation degrees were investigated to optimize the surface modification. Finally, the properties of PEGylated polymer-caged QDs were investigated, which have shown an enhancement of the colloidal and pH stability, lower cell toxicity and non-specific binding. 2. Experimental 2.1. Materials Cadmium oxide (CdO, 99.99%), trioctylphosphine oxide (TOPO, 90%), trioctylphosphine (TOP, 90%), octadecylamine (ODA, 90%), N-hydroxysuccinimide (NHS, 98%), diisopropyl carbodiimide (DIC, 99%), and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC, >98%) were purchased from Sigma–Aldrich. Sulfo-N-hydroxysuccinimide (sulfo-NHS, 98.5%) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 99%), and bis(sulfosuccinimidyl) suberate (BS3 ) were purchased from Thermo Scientific. Selenium (Se, 99.99%), Stearic acid (SA, 99%), sulfur powder (99.9%), zinc oxide (ZnO, 99%), oleic acid (OA, 99%), reduced glutathione (GSH, 90%), zinc chloride, sodium hydroxide, liquid paraffin, and ethanolamine were obtained from Sinopharm Chemical Reagent. Carboxyl-PEG-carboxyl (Mn = 2000) and aminePEG-amine (Mn = 2000) were purchased from Jiaxing Biomatrix Biotech. 2.2. Quantum dots synthesis CdSe cores were synthesized according to literature [30], and coated with ZnSe and ZnS multi-shell. Previously reported shell overcoating procedure [5] was used here to obtain QDs emitting peak at about 600 nm with quantum yield over 30% when dispersed in chloroform. Briefly, CdSe cores with first excitation peak at 564 nm were synthesized by heating a mixture of 1 mmol CdO powder, 4 mmol stearic acid and 10 ml liquid paraffin. The mixture solution was degassed and heated to 200 ◦ C with magnetic stirring until CdO was completely dissolved. After cooling to room temperature, 1 g TOPO, 3 g ODA and 10 ml liquid paraffin were added to the flask, heated to 280 ◦ C under nitrogen protection, and then 5 ml of 1 M Se in TOP was rapidly injected into the Cd-containing reaction mixture followed by cooling to room temperature after 30 min reaction at 260 ◦ C. For coating of ZnSe–ZnS shell, CdSe cores isolated by repeated precipitations from chloroform with methanol were heated to 220 ◦ C in a mixture of 5 g ODA and 15 ml liquid paraffin under nitrogen protection. Then, aliquots of Zn and Se/S precursor solutions were alternately introduced starting with Zn precursor, waiting 20 min between each of addition. After shell coating, QDs were annealed at 260 ◦ C for another 20 min, and then cooled to room temperature. Finally, core–shell QDs were isolated from solution with chloroform and methanol via centrifugation, washed three times, and stored in chloroform. 2.3. Water solublization of QDs with GSH In a typical procedure, 250 mg of GSH and 200 mg of sodium hydroxide were dissolved in 10 ml of methanol, and mixed with 5 ml of 10 ␮M of ODA coated CdSe/ZnSe/ZnS QDs solution in chloroform. After solvents in the mixture were gradually evaporated with magnetic stirring under room temperature overnight, 25 ml of water was added to disperse the precipitates. 1 ml of 1 M ZnCl2 solution was dropwise added with stirring and heated to 60 ◦ C for 10 mins to enhance the quantum yields. Then the QDs solution was filtered through a 0.22 ␮m syringe filter to remove the aggregates, and dialyzed against pure water for 48 h with a 14 kDa molecular weight cutoff (MWCO) dialysis tubing to remove the excess of

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unbound GSH. At last, GSH modified QDs aqueous solution was concentrated to about 5 ␮M by rotary evaporation under reduced pressure. 2.4. PEGylation and crosslinking of GSH–QDs During the course of our studies, different ratios of DIC, NHS, PEG and QDs were used to obtain optimal methodologies. In a typical procedure, when using GSH or amine-PEG-amine as crosslinkers, 5 ␮mol of NHS and 5 ␮mol of DIC were added into 5 nmol of GSH–QDs in 5 ml dimethylsulfoxide (DMSO) solution, and reacted for 30 min at room temperature to activate carboxyl groups on the surface of QDs. After 5 ␮mol DTT was added into the solution to quench the excess DIC [31], 2.5 ␮mol amine-PEG-amine or GSH were added to the solution, and reacted for another 4 h. The resultant mixture was dialyzed against pure water for 48 h to remove small molecules and unbound PEG with a 14 kDa MWCO dialysis tubing, then the solution was concentrated by rotary evaporation under reduced pressure. When using carboxyl-PEG-carboxyl as crosslinker, a similar procedure was employed except that carboxyl-PEG-carboxyl molecules were firstly activated with DIC, and then QDs solution added to the solution later. 2.5. Stability tests of QDs For pH stability test, QDs were incubated for 24 h in citrate buffer under acidic environment (pH 3.0–5.0), phosphate buffer saline (PBS) under neutral environment (pH 6.0–8.0) and borate buffer under basic environment (pH 9.0–11.0). For stability against crosslinker chemicals, QDs were respectively dispersed in PBS (10 mM) buffer with 5 mM EDC, 5 mM NHS, 5 mM sulfo-NHS, 0.5 mM SMCC, 0.5 mM BS3 and 0.5 mg/mL BSA. The fluorescent photos of QDs were captured with a UV light illumination after 24 h incubation in buffer, and PL intensities of QDs were recorded on the multimode microplate reader (Tecan Infinite M1000, Switzerland) with excitation wavelength at 480 nm and emission wavelength at 600 nm. Photostability studies of GSH-capped QDs and the polymer-caged QDs were conducted in PBS (10 mM) buffer under continuous ultraviolet (UV) illumination at 365 nm. The PL intensities of QDs were recorded and calculated from integration of fluorescence spectra. 2.6. Cell viability and non-specific binding of QDs NIH-3T3 and A549 cells were chosen to compare the cell viability and non-specific binding of different QDs. Approximately 5000 cells/well of cells were grown on a 96-well microplate in 180 ␮l of DMEM with 10% Fetal Bovine Serum, 50 U/ml penicillin and 50 ␮g/ml streptomycin and kept overnight at 37 ◦ C under 5% CO2 . The CdSe/ZnSe/ZnS QDs with various surface modifications were loaded in each well with a final concentration of 0, 15.6, 31.2, 62.5, 125, 250, 500 nM, and 3 duplicates were performed for each concentration. After incubation for 48 h, medium was removed and the cells were washed several times with PBS. Cell viability was determined using the sulforhodamine B (SRB) colorimetric assay. After the exposure period of 48 h, the cells were fixed with 10% cold trichloroacetic acid (TCA) for 1 h at 4 ◦ C. The TCA solution was then discarded and the cells were washed with distilled water for three times followed by complete drying. 0.2% SRB in 1% acetic acid was added to each well for 1 h at room temperature. Then the staining solution was removed, and the cells were washed with 1% acetic acid to remove excess dyes. After complete drying, the dyes were dissolved in cold 10 mM of Tris buffer (pH 10.5) followed by repeated pipetting in each well to ensure complete dissolving of dyes. The absorbance of the dye solution was measured at 540 nm

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using the microplate reader. The cell viability was calculated by normalizing with the results obtained without QDs loading. The non-specific binding of QDs with cells were investigated in A549 cells. The A549 cells were cooled to 4 ◦ C in PBS for 5 min to minimize endocytosis, and incubated with 250 nM QDs in PBS for 2 h and then washed with PBS for three times and imaged in PBS. Cells were imaged using a Zeiss Axiovert 40 CFL inverted fluorescence microscope. 2.7. Characterization Absorption (Ab) spectra of QDs were acquired with a UV–vis spectrophotometer (Shimazu 2450, Japan), and photoluminescence (PL) spectra and intensities of QDs were recorded on a fluorescence spectrometer (PerkinElmer LS-55, USA) or a multimode microplate reader (Tecan Infinite M1000, Switzerland). The fluorescence QYs of QDs were measured using Rhodamine 6G as a standard fluorophore. The concentration of QDs in water and Rhodamine 6G in ethanol were adjusted with the absorbance around 0.05 at 475 nm wavelength. Then, the PL spectra of QDs and Rhodamine 6G were taken under identical spectrometer condition with excitation wavelength at 475 nm. QY of QDs was calculated by taking a QY of 95% for Rhodamine 6G in ethanol solution [32]. The hydrodynamic diameters and size distribution of QDs were determined by a NICOMPTM ZLS system (Particle Sizing Systems 380, USA). The particle size distribution analysis was conducted by the NICOMP number-weighted distribution analysis. The transmission electron microscopy (TEM) images of QDs were captured with a Hitachi-7650 microscope operating at an accelerating voltage of 100 kV. The particle size and size distribution were performed from digital TEM images by randomly counting over 200 particles. Surface modification of QDs was characterized by gel electrophoresis. 0.5% agarose gels in Tris-EDTA acetate (TEA) buffer were run at 80 V for 20 min. Fourier transform infrared (FTIR) spectra were acquired on the FTIR spectrometer (Shimazu 8400, Japan) using KBr pellets in the range from 4000 to 400 cm−1 . Proton nuclear magnetic resonance (1 H NMR) spectra were recorded on a Bruker Avance II 400 MHz NMR spectrometer (Bruker, USA), and chemical shifts of the 1 H NMR spectra were measured in reference to internal Me4 Si (ı = 0 ppm). Thermo-gravimetric analysis (TGA) was carried out using a Mettler SDTA 851e (Mettler TOLEDO, Switzerland) instrument at heating rate of 10 ◦ C/min from 50 to 800 ◦ C with N2 as the purge gas. 3. Results and discussion 3.1. Surface modification of QDs Fig. 1 schematically illustrates strategy for achieving compact polymer-caged water soluble QDs. Firstly, highly fluorescent octadecylamine (ODA) coated core/shell CdSe/ZnSe/ZnS QDs synthesized by conventional hot injection method were transferred into water phase by replacing ODA with glutathione (GSH) on the surface of QDs, because the thiol group in GSH molecule could chelate with Zn ions on the surface of QDs. After ligands exchange process, the QDs were dispersed in water and purified by dialysis to remove excess GSH molecules. Then, GSH capped QDs were cross-linked in an intermediate polar solvent, dimethylsulfoxide (DMSO), with multi-functional molecules under activation of carbodiimide. Three different cross-linkers, including GSH, carboxyl-PEG-carboxyl and amine-PEG-amine, were selected to obtain polymer-caged QDs with different surface chemistry. Due to presence of both carboxyl and amine groups in GSH molecule, GSH on the surface of QDs can be cross-linked via the reaction

between carboxyl and amine groups with cross-linkers. After grafting one terminal of the bifunctional PEG onto surface of QDs, the other terminal of PEG could randomly react with neighbor GSH molecule or just pend around nanoparticles. Glutathione as one of the most abundant small molecules in biosphere, is serving to detoxication of xenobiotics and heavy metals, especially those having high affinity to the thiol groups [33,34]. It is reported highly fluorescent QDs were directly synthesized in aqueous phase using GSH as capping agent [35,36]. Here, water solubilization of organic soluble CdSe/ZnSe/ZnS QDs were performed by displacing the native hydrophobic ligands with thiol groups in GSH, which can binding with Zn ions on the surface of QDs. Complete ligands exchange were confirmed by 1 H NMR spectroscopy and FTIR spectroscopy. As shown in Fig. 2c, before the ligands exchange, the proton signals of ODA moieties were clearly observed, and the chemical shift in 1.25 ppm is attributed to protons originated from CH2 segment in ODA molecules. In the FTIR spectrum of ODA-coated QDs, as shown in Fig. 2b, the characteristic IR peaks at 2920 and 2850 cm−1 originated from the stretching vibrations of CH2 in ODA. In contrast, GSH modified QDs (GHS–QDs) displayed a significantly different pattern of the proton signals: at 3.86 and 2.51 ppm, which correspond to the GSH ligands. Both 1 H NMR and FTIR were confirmed the total ligands exchange on the surface of QDs and no obvious ODA molecules were detected in GSH–QDs. From the thermo-gravity analysis, as shown in Fig. S4, decomposition of original ODA–QDs began from 175 ◦ C with a weight loss ca. 41.4% at 800 ◦ C, whereas GSH–QDs decomposed from 240 ◦ C with a weight loss ca. 13.2% at 800 ◦ C, which could also reflect that ODA molecules on the surface of original QDs were replaced by GSH molecules. As shown in Fig. 2a, the absorption and PL spectra of QDs before and after ligands exchange were compared. The peak position had only a slight red shift (less than 5 nm), and the full width at halfmaximum (FWHM) of GSH–QDs were also slight broadened (less than 5 nm), which may be due to the formation of an additional thin ZnS shell and not uniform shell thickness on the surface of QDs. The ZnS shell formation may attribute to the reaction between the thiol groups in GSH and Zn ions provided by zinc chloride [37]. The insert photos in Fig. 2a indicate that the GSH–QDs are highly hydrophilic and stably dispersed in water. The GSH–QDs solution was clearly transparent, and no appearance of aggregates was observed. Even shaking with chloroform, the GSH–QDs water solution quickly finished phase separation in seconds, and no agglomeration was observed, which indicated the bind between GSH and QDs were stable and irreversible by mixing with chloroform. The hydrodynamic diameter of GSH–QDs is 7.5 ± 0.5 nm determined from the dynamic light scattering measurement, which indicate the GSH–QDs are compact and monodispersed, and the diameters of QDs are not significantly increased compared with hydrophobic QDs, which have an average diameter 5.7 ± 0.5 nm measured from TEM images (Supporting information Fig. S1). The quantum yield of QDs was only slightly reduced from 32% in chloroform to 26% in water, and the product yield could reach over 80%. However, small molecules are prone to detach from the surface of QDs, which would lead to aggregation and quantum yield decreasing of QDs [25]. The phytochelatin-related polymer-caged coating by crosslinking of GSH molecules on the surface of QDs could prevent desorption of capping ligands from QDs, and improve the stability of QDs [25,38]. PEGylation of inorganic nanoparticles could increase the colloidal stability, hydrophilicity and biocompatibility [28,29]. Thus, bifunctional PEG molecules, which could serve as both cross-linkers and PEGylation reagent, were selected for crosslinking GSH molecules on the surface of QDs. Polymercaged QDs were achieved by reaction between carboxyl and amine groups under the activation of carbodiimide.

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Fig. 1. Schematic illustration of the surface modification of octadecylamine (ODA)-coated organic soluble QDs. The organic soluble QDs were water solubilized through replacing ODA molecules on the surface of QDs by glutathione (GSH) molecules through thiol groups chelating with metal cation (Zn2+ or Cd2+ ). Then GSH molecules on the surface of QDs, which contain both carboxyl and amine groups, were cross-linked with the bifunctional PEG (carboxyl-PEG-carboxyl or amine-PEG-amine) in the presence of carbodiimide.

Compared with the GSH capped QDs, the optical properties of polymer-caged QDs did not significantly change after crosslinking process. The absorbance and PL spectra of polymer-caged QDs are similar to GSH–QDs (Supporting information Fig. S2), which indicated that the surface crosslinking process has negligible influence on the spectra properties of QDs, including peak position, FWHM and spectra shape. QYs of polymer-caged QDs crosslinked with GSH and carboxyl-PEG-carboxyl have a slight reduction, and QYs of QDs with amine-PEG-amine crosslinkers have a moderated reduction. Additional higher amine-PEG-amine functional densities also lead to more reduction of QYs. The product yields of crosslinking GSH coated QDs can achieve over 80%, and the loss of QDs in the process is negligible. Thus, this surface modification method is potential for the large-scale preparation of polymer-caged QDs. Hydrodynamic diameters of QDs before and after crosslinking process were determined by the dynamic light scattering. As shown in Fig. 3a, the diameters of QDs after crosslinking process respectively increased from 7.5 ± 0.5 nm to 9.7 ± 1.3 nm, 14.6 ± 1.6 nm and 12.8 ± 1.1 nm for polymer-caged QDs with GSH, carboxyl-PEGcarboxyl and amine-PEG-amine as crosslinkers. As shown in Figs. 3b and 4, the relative sizes of QDs were also observed by gel electrophoresis, on which different size of QDs has different relative migration distances. The increase of hydrodynamic diameter is an evidence for the formation of a polymer-caged shell surrounding the QDs surface, and larger diameter of QDs modified with PEG crosslinkers indicated that PEG molecule were attached onto the

surface of QDs. The narrow size distributions in DLS measurements and narrow width of bands observed in gel reflect the narrow size distribution of the GSH–QDs and polymer-caged QDs. The size distributions of QDs after surface crosslinking process were not obviously broadened indicated that the crosslinking reaction was mainly occurred on the surface of single QDs and not among interparticles. The grafted PEG molecules can also be confirmed from FTIR spectra, as shown in Fig. S3. The absorption peaks around 1100 cm−1 in the PEGylated polymer-caged QDs were assigned to those of C O C bonds in PEG. From the thermo-gravity analysis, as shown in Fig. S4, compared with cGSH–QDs, more weight loss of PEGylated polymer-caged QDs indicated that PEG molecules were linked on the surface of QDs. Ratios of PEG crosslinkers, NHS and DIC were varied to determine the optimal condition for crosslinking surface ligands with less decreasing of quantum yields and colloidal stability of QDs. The ratio 2000:4000:4000 of PEG/DIC/NHS equivalent per quantum dot exhibited the best colloidal stability for carboxyl–PEG–carboxyl as crosslinkers, and the optimal ratio for amine-PEG-amine crosslinkers is 1000:2000:2000 of PEG/DIC/NHS equivalents per quantum dot. The amount of PEG cross-linkers were varied to obtain improved colloidal stability and monodispersity of nanoparticles. As shown in Fig. 4, the migration distances of QDs gradually decreased with increasing proportion of PEG cross-linkers. However, when the ratio of PEG cross-linkers to nanoparticle equivalent to 2000:1, the migration distance of QDs increased, which may

Fig. 2. (a) Normalized absorbance and PL spectra of CdSe/ZnSe/ZnS QDs in chloroform capped with ODA and in water capped with GSH. Inserted photos are a visual comparison of QDs before (left) and after (right) ligand exchange with GSH under a UV lamp excitation at 365 nm. (b) FTIR spectra of ODA and GSH capped QDs. (c) 1 H NMR spectra of ODA capped QDs (CDCl3 ) and GSH capped QDs (DMSO-D6).

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Fig. 3. DLS and gel electrophoresis of aqueous CdSe/ZnSe/ZnS QDs with different surface chemistry. (a) DLS particle size distribution and (b) gel electrophoresis of (1) GSH–QDs, (2) cGSH–QDs, (3) COOH-PEG-QDs (PEG:QDs = 2000:1), (4) NH2 -PEG-QDs (PEG:QDs = 2000:1). The hydrodynamic diameter of nanoparticles was measure to be (1) 7.5 ± 0.5 nm, (2) 9.7 ± 1.3 nm, (3) 14.6 ± 1.6 nm, (4) 12.8 ± 1.1 nm from DLS. QDs used for agarose gel electrophoresis analysis were already stored at 4 ◦ C for over 3 months.

attribute to PEG coating on the surface of QDs is most compact and highest density under this crosslinking condition. Continuing increasing the proportion of PEG crosslinkers to QDs would lead to broadening the bands width of QDs in gel electrophoresis, which may caused by uneven grafting of PEG molecules onto QDs or the inter-particles crosslinking of QDs at higher crosslinkers concentration. 3.2. Colloidal and pH stability of QDs The colloidal and PL stability of QDs with different surface chemistry was investigated by dispersing QDs in pH buffer with pH value ranging from 3.0 to 11.0. As shown in Fig. 5, the small molecule capped GSH–QDs were colloidal stable in the buffer with pH

Fig. 4. Agarose gel (0.5%) electrophoresis of QDs with different surface chemistry, GSH–QDs (1), carboxyl-PEG-carboxyl (a) and amine-PEG-amine (b) cross-linkers with the proportion of PEG molecules per nanoparticle equal to 500, 1000, 2000, 4000 (2–5). The gels were run at 80 V for 20 min.

ranging from 4.0 to 10.0, but precipitated in more acidic and basic buffer. The crosslinked cGSH–QDs had shown improved colloidal stability in the basic buffer, however lost the stability in the acidic buff at pH 3.0 and 4.0. The better colloidal stabilities of PEGylated polymer-caged QDs were obviously observed in more corrosive buffer, and no precipitates of QDs were observed in the buffer with pH ranging from 3.0 to 11.0. Moreover, the PL intensities of polymer-caged QDs had fewer fluctuations with pH values of buffer, and this make QDs are suitable for biological application in a wide pH range of buffer. Nanoparticles in solution are mainly stabilized by electrostatic and steric repulsion. GSH molecule with both NH2 and COOH displays zwitterionic nature with pKa ’s in both acidic and basic environment, and thus could be ionized in a broad pH range and have less pH responsive properties [39–42]. However, the strong acidic condition could etching of QDs or destabilized the ligand-QDs bonds, thus lead to the reduction of PL intensities and precipitation of nanoparticles. Grafting of PEG molecules onto QDs could increase water solubilization of QDs due to the hydrophilic nature PEG backbones, and increase the steric repulsion between nanoparticles. Thus, the PEGylated polymer-caged QDs have shown colloidal stability in a broader pH range than GSH–QDs and cGSH–QDs. The photostability of water soluble QDs in PBS buffer was investigated as a function of UV irradiation time at room temperature As shown in Fig. S5, the fluorescence intensities of QDs have an enhancement at the beginning due to UV irritation [43], and have a slight decrease in 1 h. Polymer-caged QDs are more resistant to photobleaching than GHS–QDs, which can be attributed to polymer-caged surfactants are not easy detached from the surface of nanoparticles than the small molecules. For biological application, QDs were usually conjugated with biomolecules, such as proteins, DNA and peptides, through some crosslinker chemicals, and thus it is important to maintain the colloidal and PL stability in such chemicals solution. As shown in Fig. 6, compared with GSH–QDs and cGSH–QDs, the PEGylated polymercaged QDs have remarkable stability. The crosslinked cGSH–QDs were easy to precipitate in all the crosslinking chemicals solution, and GSH–QDs were precipitated in EDC buffer. This chemical stability could facilitate conjugation of biomolecules onto QDs and maintaining the PL and colloidal stability of QDs in the conjugation process, which is important for the further use of QDs bioconjugation.

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Fig. 5. pH stability comparison of different surface chemistry QDs in pH buffer with pH ranging from 3.0 to 11.0.

3.3. Non-specific binding and cytotoxicity An absence of non-specific binding is essential to reliable targeting and sensing application of QDs. To investigate the non-specific binding of polymer caged QDs, we incubated A549 cells with cGSH–QDs and COOH-PEG-QDs at high concentrations (250 nM) in order to highlight the differences between the coatings. Fig. 7

shows bright field (left) and fluorescence (right) images of fixed A549 cells after washing with PBS. As expected, the cGSH–QDs incubated cells shows a high level of non-specific binding to the cells as seen by the strong fluorescence associated with the cells. Conversely, the COOH-PEG-QDs sample shows negligible non-specific binding as seen by minimal cellular associated fluorescence. This result is in agreement with previous reports which have found that

Fig. 6. Stability comparison of different surface chemistry QDs in crosslinking chemicals solution, 5 mM EDC, 5 mM NHS, 5 mM sulfo-NHS, 0.5 mM SMCC, 0.5 mM BS3 , 0.5 mg/mL BSA in PBS (pH 7.4, 10 mM) buffer.

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Fig. 7. Representative images of nonspecific binding of QDs to A549 cells with incubation at 250 nM QDs concentration for 2 h at 4 ◦ C followed by 3 wash with PBS buffer before imaging.

PEG coated QDs can significantly reduce the non-specific binding of QDs to cells. The low cytotoxicity of QDs is also one of the major requirements in application for vivo imaging and drug delivery. We further evaluated the cytotoxicity of cGSH–QDs, COOH-PEG-QDs and NH2 PEG-QDs in both NIH-3T3 and A549 cells. As shown in Fig. 8, below the concentration of 31.25 nM (measured with UV absorption at first extinction peak of QDs), cell viabilities after incubation with QDs for 48 h are above 90%, and the QDs with different surface modification have negligible difference at low concentration. At higher QDs concentration, PEGylated QDs show lower cytotoxicity compared with cGSH–QDs, and NH2 -PEG-QDs have lower cytotoxicity than COOH-PEG-QDs. 4. Conclusions In summary, we have prepared compact PEGylated polymercaged QDs with small hydrodynamic size, high quantum yields, good colloidal stability, low non-specific binding to cells and low cytotoxicity. Polymerization of capping ligands on the surface of QDs with bifunctional PEG crosslinkers combined PEGylation and crosslinking in one step. This strategy is potential for large amount preparation of water soluble compact polymer-caged QDs, and could also be applied to other peptide-coated nanoparticles to achieve PEGylation and polymer-caged coating which could improve the colloidal stability and biocompatibility of nanoparticles. Acknowledgments Fig. 8. Cytotoxicities assay of cGSH–QDs, COOH-PEG-QDs and NH2 -PEG-QDs at different concentrations in A549 (a) and NIH-3T3 (b) cells for 48 h incubation.

The authors thank Yongjian Yang and Xia Ling at Shanghai Institute for Food and Drug Control (SIFDC) for assistance in the PL spectra measurements, Yi Hu, Kun Li and Yuting Sun for cells culture, and Huimin Hu and Ying Tang for assistance with TEM

P. Zhang, H. Han / Colloids and Surfaces A: Physicochem. Eng. Aspects 402 (2012) 72–79

analysis. This work was supported by funds from Shanghai STCSM Project (no. 09441900700).

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Appendix A. Supplementary data [22]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfa.2012.03.022.

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