Surfactant-assisted synthesis of water-soluble and biocompatible semiconductor quantum dot micelles

Surfactant-Assisted Synthesis of

Water-Soluble and Biocompatible

Semiconductor Quantum Dot Micelles

Hongyou Fan,*,?,?Erik W.Leve,?Chessa Scullin,§John Gabaldon,?David Tallant,?

Scott Bunge,?,|Tim Boyle,?Michael C.Wilson,§and C.Jeffrey Brinker?,?

Chemical Synthesis and Nanomaterials Department,Sandia National Laboratories,

Albuquerque,New Mexico87106,The Uni V ersity of New Mexico/NSF Center of

Micro-Engineered Materials,Department of Chemical and Nuclear Engineering,

Albuquerque,New Mexico87131,and Department of Neuroscience,School of

Medicine,The Uni V ersity of New Mexico,Albuquerque,New Mexico87106

Received January4,2005;Revised Manuscript Received February4,2005

ABSTRACT

We report a simple,rapid approach to synthesize water-soluble and biocompatible fluorescent quantum dot(QD)micelles by encapsulation of monodisperse,hydrophobic QDs within surfactant/lipid micelles.Analyses of UV?vis and photo luminescence spectra,along with transmission electron microscopy,indicate that the water-soluble semiconductor QD micelles are monodisperse and retain the optical properties of the original hydrophobic QDs.The QD micelles were shown to be biocompatible and exhibited little or no aggregation when taken up by cultured rat hippocampal neurons.

Semiconductor quantum dots(QDs)with superior optical properties are a promising alternative to organic dyes for fluorescence-based bioapplications.The potential benefits and use of conjugated QD probes to monitor cellular functions has prompted extensive efforts to develop methods to synthesize water-soluble and biocompatible QDs that can be widely used for fluorescent-based bioimaging.1-11In general, the key to develop QDs as a tool in biological systems is to achieve water solubility,biocompatibility and photostability, and,importantly,to provide flexible QD surface chemistry/ functionality that will enable efficient coupling of these fluorescent inorganic probes to reagents capable of targeting and/or sensing ongoing biological processes.

We recently developed a method to prepare gold nano-crystal(NC)micelles in aqueous media.12The method is to incorporate monosized,hydrophobic gold NCs into the hydrophobic interiors of surfactant micelles.The uniform gold NC size and self-assembled monolayer-like nature of its surface resulted in the formation of monosized gold NC micelles composed of a gold NC core and a hybrid bilayer shell with precisely defined primary and secondary layer thicknesses.Here,we extend this technique to the synthesis of water-soluble,biocompatible semiconductor QD micelles. This synthetic approach avoids complicated multistep pro-cedures1,6and promises to add to the versatility of QD-based probes for investigating intracellular transport and other cellular signaling pathways in living cells. Monodisperse CdSe and CdSe/CdS core/shell QDs were synthesized according to previous methods13,14using trio-ctylphosphine(TO)and oleylamine(OA)as stabilizing agents.In a typical QD micelle synthesis procedure,a concentrated suspension of QDs in chloroform is added to an aqueous solution containing a mixture of surfactants or phospholipids with different functional headgroups such as ethylene glycol(-PEG)and amine(-NH2).PEG is used to improve biocompatibility and amine groups provide sites for bioconjugation.Addition of the QD chloroform suspension into the surfactant/lipid aqueous solution under vigorous stirring results in the formation of an oil-in-water micro-emulsion.15Evaporation of chloroform during heating(40-80°C,～10min)transfers the QDs into the aqueous phase by an interfacial process driven by the hydrophobic van der Waals interactions between the primary alkane of the stabilizing ligand and the secondary alkane of the surfactant, resulting in thermodynamically defined interdigitated bilayer structures(Scheme1d).Based on evaluation of water solubility and stability using ultraviolet-visible spectroscopy

*Corresponding author.E-mail:hfan@https://www.sodocs.net/doc/d712935404.html,;Tel:505-272-7128; Fax:505-272-7336.

?Sandia National Laboratories.

?The University of New Mexico/NSF Center of Micro-Engineered Materials.

§The University of New Mexico,Department of Neuroscience,School of Medicine.

|Present address:Department of Chemistry,Kent State University,P.O. Box5190,Kent,OH44242-0001.

NANO LETTERS

2005 Vol.5,No.4 645-648

(UV -vis),transmission electron micrograph (TEM),and fluorescent spectra,optimized lipids were those with at least 6-8carbons in their hydrophobic alkane chains.

QD-micelle solutions exhibited the same visible and emission colors as hydrophobic QDs,as shown in the optical micrograph (Figure 1A).The formation of individual CdSe/CdS QD micelles was further charaterized by UV -vis spectroscopy shown in Figure 1B.We observed no difference in the position or width of the absorbance bands at ～480

nm and ～590nm from hydrophobic QDs and QD micelles,which suggests that QD micelles maintain their optical properties in water.Formation of ordered hexagonal close packing shown in TEM (Figure 1C),as expected for individual monosized QDs,further confirmed the mono-dispersity of QD micelles.Photoluminescence (PL)spectra of OA-stabilized hydrophobic QD and QD micelles prepared using 1,2-dioctanoyl-sn -glycero-3-phosphocholine (C8-lipids)are shown in Figure 1D.PL of QD micelles exhibited no obvious shift in emission wavelength.Studies of photosta-bility of these water-soluble NC micelles under long-time laser irradiation showed that these QD micelles exhibited no loss of PL intensity in water (Figure 2A inset).The stability of QD micelles in a cellular aqueous environment was confirmed by incubation with rat hippocampal neurons (see below).

The PL persistence (lifetimes)from OA-stabilized CdSe/CdS in toluene,CdSe/CdS-micelle water solutions,and Rhodamine B (RB)in ethanol were measured and calculated according to Donega et al.16and are shown in Figure 2A.The persistence was measured during pulsed,450-nm excita-tion by monitoring emission at the peak of the PL profile.The RB persistence is only slightly longer than the instru-mental bandwidth.The log e plots of the persistence curves from the CdSe QDs in toluene and H 2O/C8lipid solutions were similar and nearly linear,with a (1/e)relaxation constant of ～30nanoseconds,which was much longer than the lifetime of RB (～5nanoseconds).

The advantage of our method to provide water-soluble QD micelles is its capability to utilize a wide variety of surfactants/lipids with different functionally terminated groups,which can be used to improve biocompatibility and to enable bioconjugation.The encapsulation is accomplished in a single,rapid (10minute)step of solvent evaporation.In our method,the stabilization of hydrophobic QDs in water relies on the thermodynamically favorable interdigitation of sur-factants and QD stabilizing ligands formed after solvent

Scheme 1.Formation of Water-soluble QD-micelles through

Incorporation in Surfactant or Phospholipid Micelle

a

a

Legend: a.An OA-stabilized CdSe/CdS QD;b and c.OA-stabilized CdSe/CdS QDs are encapsulated inside surfactant/lipid micelles using an oil-in-water microemulsion technique where evaporation of oil drives interfacially mediated QD transfer into water-soluble micelles.12d.Thermodynamically defined interdigi-tated “bilayer”structure stabilized QDs in

water.

Figure 1.(A)Optical micrograph of CdSe QDs in chloroform and CdSe micelles in water prepared using cetyltrimethylammonium bromide and imaged under room light and 350-nm UV light.(B)UV -vis spectra of (1)CdSe/CdS in hexane,(2)CdSe/CdS micelles prepared in phosphate buffer solution by using 1,2-dioctanoyl-sn -glycero-3-phosphocholine (C8-lipid)and hexadecylamine (HDA),and (3)1,2-distearoyl-sn -glycero-3-phosphoethanolamine-N -amino (polyethylene glycol)(aPEG)and dipalmitoyl phosphatidylcholine (DPPC).(C)TEM image of CdSe/CdS micelles deposited on TEM grid from a drop of CdSe/CdS micelle water solution.(D)Photoluminescence spectra of CdSe/CdS in toluene (solid line)and C8lipid encapsulated CdSe/CdS micelles in water (dashed

line).

Figure 2.(A)Persistence curves from CdSe/CdSe in toluene,RB in ethanol,and CdSe/CdS micelles in water solution prepared by using C8-lipid.(Inset)PL intensity CdSe/CdS micelles in water solution vs laser irradiation time.

evaporation,enabling rapid efficient transfer of the QDs into the aqueous phase.The QD micelles preserve the optical properties of the original source QDs,such as PL intensity and lifetime etc.,as the encapsulation process involves no chemical substitution reactions.In contrast,in other methods, such as the ligand exchange method,chemical substitution occurs at the QD interface.This usually results in QD aggregation and changes in QD physical properties.17,18 Additionally,our method can effectively prevent QD micelles from aggregation due to the interdigitated“bilayer.”

To evaluate the biocompatibility of CdSe/CdS QD mi-celles,we performed an initial study to examine their uptake in cultured rat hippocampal neurons.While the route through which QDs are taken up by cells has yet to be resolved fully, they do appear to accumulate in intracellular vesicular compartments19-21in several cell types,suggesting uptake may be mediated,at least in part,through endocytosis. Because synaptic vesicle recycling is tightly coupled to neurotransmitter release,22endocytosis can be manipulated by depolarization of the plasma membrane to excite neurons, evoking exocytosis for neurotransmitter release followed by endocytotic retrieval of synaptic vesicle membrane from nerve terminals.As shown in Figure3A,QD micelles (emission592nm,red)accumulate in neurons,distinguished by immmunohistochemical counterstain for NeuN(green), after exposure for16h in basal serum-free growth media.

A three-dimensional reconstruction of confocal images confirmed the intracellular localization of QD fluorescence after long-term exposure(see Supporting Information,movie 1).The observation that QDs retain their fluorescence character after a fairly long incubation suggests that QDs can accumulate without aggregation and a general compat-ibility of the micelle coating within a cellular environment. To obtain a more detailed view of QD uptake,we used pulse-chase labeling to expose hippocampal cultures to QD micelles during membrane depolarization(induced by ad-dition of55mM KCl)to trigger neurotransmitter release and coupled exo/endocytosis,followed by a return to basal media. After a10min exposure to QDs during depolarization,robust QD fluorescence was detected by confocal microscopy in a widespread,yet clustered pattern surrounding NeuN-positive neurons Figure3,panel B),consistent with a diffuse,punctate localization and uptake in neuronal processes and synapses. Interestingly,following exchange for basal media without QDs,there was an apparent time-dependent increase in the perinuclear accumulation of QD fluorescence accompanied by a decrease in the more widespread distribution seen immediately after KCl depolarization(Figure3,Panels C, D).By5h,the majority of QD fluorescence appeared to locate to a perinuclear compartment adjacent to NeuN stained neuronal nuclei.The results indicate that under these conditions QDs were effectively taken up by an endocytotic mechanism that was promoted by increased synaptic vesicle recycling and potentially followed by transport to lysosomes. Trafficking of QDs to lysosomal compartments may reflect

autophagy,a vesicular-mediated process that underlies catabolic recycling and degradation of macromolecules, including both cellular and xenotypic agents,such as bacteria and viral pathogens.23Autophagy may be a particularly critical mechanism for maintaining the cellular integrity of neurons,which are long-lived and generally show little

or Figure3.Images of CdSe/CdS micelles,prepared using modified phospholipids including aPEG,DPPC,and HDA,in cultured rat hippocampal neurons.In this case where aPEG and DPPC are less water soluble,aPEG and DPPC were directly added into QD chloroform solution and water was added after evaporation of chloroform to prepare a QD micelle aqueous solution.Dispersed cultures of hippocampal neurons,prepared from3day old rat pups and grown on polylysine/collegen coated glass coverslips for two weeks in Neurobasal A media with B-27supplement(Invitrogen/ GIBCO),were exposed to QD micelles emitting at592nm.After incubation,the cells were fixed in4%paraformaldehyde/4% sucrose,permeabilized and stained with antibodies to neuronal specific marker NeuN(Chemicon,Temecula,CA),followed by FITC(Panel A)or Alexa Fluor488(Panel B-D)labeled antimouse secondary antibody(Molecular Probes,Eugene OR).Panel A, fluorescent microscopy image of QD accumulation(red,arrows) after16h of incubation(～1mg/ml QD micelles)in neurons identified by nuclear staining for NeuN(green).Panels B-D, confocal microscopy of pulse-chase labeling with QD micelles. Incubation with QD micelles during depolarization with55mM KCl for10min(panel B)shows dispersed,punctate staining (indicated by astericks)characteristic of neurite processes;whereas after chase in basal media without QD micelles for1h(panel C) or5h(panel D)shows increasing perinuclear localization(arrows) with a corresponding decrease in the punctate stain seen in the10 min labeling.Scale bar,10microns,for panels B-D.Images of1μm thick optical sections were obtained with a Zeiss Meta System Confocal Microscope using a40×objective.QDs were imaged using405mm excitation and560-615nm emission;Alexa488 NeuN staining with488nm excitation and505-530nm emission.

no turnover in most regions of the nervous system,and could play a role in neuronal cell death under certain conditions.24 The ability to use QDs to probe the trafficking of autophagic vacuoles promises to provide a useful tool to track this cellular process,but whether this tool itself elicits an autophagic response,precipitating cell death over time,will require further study.

In conclusion,we report a simple and rapid synthesis of water-soluble QD micelles through encapsulation of hydro-phobic QDs into surfactant/lipid micelles.The resulting water-soluble QD micelles retain the optical properties of the original hydrophobic QDs.In addition to bioapplications, the water-soluble QD micelles are suitable building blocks for further self-assembly into ordered arrays in inorganic matrices needed for enhanced optical properties and for achieving mechanical robustness in nanodevices.25,26 Acknowledgment.We thank Drs.John Guzowski and Teiko Miyashita for training and help with fluorescent microscopy.This work was partially supported by the U.S. Department of Energy(DOE)Basic Energy Sciences Pro-gram,Sandia National Laboratory’s Laboratory Directed R&D program,the Air Force Office of Scientific Research, NSF/UNM Science and Learning Center(SLC)Catalyst/ Planning Grant,and Center for Integrated Nanotechnologies (CINT).TEM studies were performed in the Department of Earth and Planetary at University of New Mexico.Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company,for the United States Depart-ment of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.

Supporting Information Available:Confocal images of QD accumulation.This material is available free of charge via the Internet at https://www.sodocs.net/doc/d712935404.html,.

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