Hydrophobin-Encapsulated Quantum Dots - ACS Applied Materials

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Hydrophobin-Encapsulated Quantum Dots Shohei Taniguchi, Lydia Sandiford, Maggie S Cooper, Elena Rosca, Raha Ahmad Khanbeigi, Simon Michael Fairclough, Maya Thanou, Lea Ann Dailey, Wendel Wohlleben, Bernhard von Vacano, Rafael T. M. de Rosales, Peter Dobson, Dylan Owen, and Mark A Green ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11354 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 7, 2016

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ACS Applied Materials & Interfaces

Title: Hydrophobin-Encapsulated Quantum Dots Authors: Shohei Taniguchi,a Lydia Sandiford,b Maggie Cooper,b Elena V. Rosca,c Raha Ahmad Khanbeigi,c Simon M. Fairclough,a Maya Thanou,c Lea Ann Dailey,c Wendel Wohlleben,d Bernhard von Vacano,d Rafael T. M. de Rosales,b Peter J. Dobson,e Dylan M. Owen,a Mark Green.a* a) Department of Physics, King’s College London, Strand, London WC2R 2LS, UK. b) Department of Imaging Chemistry and Biology, Division of Imaging Science and Biomedical Engineering, King’s College London, 4th floor, Lambeth Wing, St Thomas’ Hospital, London SE1 7EH, UK. c) Institute of Pharmaceutical Science, King's College London, 5th Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. d) Material Physics Research, BASF SE, 67056 Ludwigshafen, Germany. e) Warwick Manufacturing Group, International Manufacturing Centre, University of Warwick, Coventry, CV4 7AL, UK. *corresponding author [email protected] Keywords; quantum dots, amphiphilic, phase transfer, proteins, conjugation, imaging,

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Abstract The phase transfer of quantum dots to water is an important aspect of preparing nanomaterials that are suitable for biological applications, and although numerous reports describe ligand exchange, very few describe efficient ligand encapsulation techniques. In this report, we not only report a new method of phase transferring QDs using a amphiphilic protein (hydrophobin), we describe the advantage of using a biological molecule with available functional groups, their use in imaging cancer cells in vivo and other imaging applications.

Introduction The surface chemistry of quantum dots is a key parameter when considering real-life applications and one must choose the correct passivating agent to ensure the optical and electronic properties of the core material are maintained. In the biological applications of quantum dots, the surface ligands are the singular most important factor in obtaining useful materials (once the optical features of the quantum dots have been preserved).1 Usually, quantum dots designed for biological imaging are synthesised by organometallic chemistry allowing materials with specific emissive properties to be prepared.2 This means the particles are often insoluble in water and require phase-transfer using novel surface chemistries before finding a practical use. The desired features of transfer surface ligands include the maintenance of the emissive properties, induction of water-solubility, and if needed, the introduction of a functional group to allow targeted delivery or conjugation. In general, there are two routes to ensuring these properties are met; surface encapsulation or surfactant exchange.1,3 In surfactant exchange, the original hydrophobic surface ligands are removed and replaced with hydrophilic alternatives. This might be considered the most common method of phasetransfer with many ligands developed to provide the desired attributes. In this case, a relevant functional group to coordinate to the particle surface is required, whilst the rest of the molecule can provide water solubility and further functional groups if needed. Many simple species, such as amino acids can be used to phase-transfer quantum dots and simple biological molecules, such as thiolated DNA4 and cysteine5 are often used to great effect. Whilst this is a simple and effective route to phase-transfer, it is often accompanied with a substantial reduction in the optical properties of the quantum dot. With surface encapsulation, the entire particle (including the original surface ligand) is enveloped by a new amphiphilic species, usually by the interdigitation of hydrophobic ligands, providing a new hydrophilic surface. This species is usually an engineered polymer6 or lipid7 and can have functionalities included, providing conjugation points for further attachments.8-11 This technique is generally less detrimental to the optical properties, but increases the hydrodynamic diameter of the particle. Both routes are routinely used for a variety of inorganic nanoparticles, including metals and magnetic metal oxides.


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Whilst the exchange method has seen hundreds of molecules employed, the encapsulation method has not expanded at the same rate presumably due to the small number of suitable amphiphilic molecules available, with very few biological molecules employed to encapsulate particles. Here, we report the use of hydrophobins (low molecular weight amphiphilic proteins) as a new biological encapsulation reagent to phase-transfer inorganic nanoparticles and their use in biological imaging (figure 1).

Figure 1 - Cartoon showing the tertiary protein structure, its use in phase transfer and photos of quantum dots before and after phase transfer from toluene to water. Although quantum dot-protein conjugates have previously been reported as bridges to other functional biological molecules,12,13 this is the first time an actual protein has been used in encapsulation and phase transfer. Hydrophobins are small (ca. 1.2 nm diameter) cysteine-rich amphiphilic proteins consisting of approximately 100 amino acid residues. Although the protein sequence differs between originating organisms, hydrophobins typically have eight cysteine residues (four disulfide groups) in a specific 3 ACS Paragon Plus Environment


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pattern making a hydrophilic region which contributes to the hydrophobin’s specific characteristics and structural robustness,14 whilst aliphatic side-chains in the loop region of the protein result in a hydrophobic patch, yielding an amphiphilic structure. Hydrophobins are of interest due to their potential biocompatibility as well as providing stability against pH and ionic salts arising in biological media.15 Other groups have exploited the phase transfer potential provided by hydrophobins, notably with singlewalled carbon nanotubes and gold nanoparticles forming bioconjugates,16 graphene sheets17 and silicon particles. Whilst hydrophobins have traditionally been difficult to obtain, new advances in recombinant processing have made hydrophobins a realistic option for use in phase transfer. In this study, we used commercially-available H*protein B (class-I hydrophobin), a recombinant protein consisting of hydrophobin DewA (A. nidulans), 40 N-terminal amino acids of yaaD with a molar mass of 18.8 kDa,18 to phase transfer semiconductor quantum dots to aqueous solution and then demonstrated the resulting composite’s use in various imaging applications.

Results and Discussions The hydrophobins used had a diameter of ca. 1.2 nm as determined by dynamic light scattering (DLS, Figure S1). The initial diameter observed (15 nm) disappeared after dilution, whereas the peak observed at ca. 1.5 nm increased. This behaviour can be explained as self-assembled hydrophobins exhibited agglomerated structures at the beginning of dilution, which gradually dis-assembled to lower sized monomeric structures. The final hydrodynamic diameter calculated from DLS result was 1.2 nm ± 0.2 nm, which is a close value to the hydrodynamic diameter of H*Protein B diluted 100 times and stored at 4 ○C over a day (1.3 nm ± 0.3 nm). As such, prior to phase-transfer, the protein solution was diluted in water and left standing to prevent self-assembly of the protein. To this was added a non-polar organic solution of particles during sonication, producing a cloudy emulsion. After a brief storage period and filtration, centrifugal concentration and further dilution with water, a transparent aqueous solution of particles was obtained. We suggest the amphiphilic nature of the protein was exploited by inter-digitating the aliphatic chain of the protein with the hydrophobic capping agent of the quantum dot, leaving the cysteine-rich hydrophilic region exposed to solution, making the composite water-soluble. Hydrophobins are known to be robust and it is unlikely that sonication affected the structure. For example, it is suggested that class II hydrophobins keep their secondary and ternary structure after exposure to sodium dodecylsulphate, a compound routinely used to denature proteins.19 In addition, ultrasound treatment has been applied in some phase transfer reactions, showing no evidence of protein unfolding.16 Quantum dots transferred to water displayed no significant shift in either absorption or emission wavelengths (a typical example is shown in figure 2).


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Figure 2 – absorption and emission spectra of CdSeS/ZnS quantum dots in toluene, before phase transfer (black line) and after phase transfer with hydrophobins (dotted grey lines). Inset; photograph of QD/hydrophobin solution, photoexcited at 365 nm in the dark. Typically, emission quantum yields reduced, to a maximum of 50 % (for example from ca. 60 % to ca. 30 %) although this was dependant upon particle type, quality, and shell thickness. Despite this reduction, the solution still emitted brightly under 365 nm excitation (figure 2, inset). To test the stability, sealed solution of CdSeS/ZnS quantum dots were monitored over a one-week period (figure 3). The crude solution of quantum dots in hexane showed a gradual increase in emission intensity, commonly refereed to as photo-brightening. The simple hydrophobin-capped particles displayed a drop in emission intensity of about 10 % over a 7-day period, whilst the cross-linked hydrophobin-capped particles lost approximately 50 % of their emission intensity over a 2-day period, exhibiting only ca. 10 % of their original emission after one week.



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Figure 3 – emission intensity of hexane (crude) solution of quantum dots (black squares), hydrophobin-capped (red circles) and hydrophobin-capped and glutaraldehyde cross-linked quantum dots (green triangles) over a one-week period. Electron microscopy showed the quantum dots remained as discrete individual particles (figure 4). DLS suggested that CdSeS/ZnS particles, ca. 5 nm in diameter as determined by electron microscopy had an initial hydrodynamic diameter of up to ca. 9 nm once coated, although diameters of up to ca. 60 nm were occasionally observed, attributed to clustering in solution or free protein agglomerates. The zeta potential of the hydrophobin-capped quantum dots was -42.8 mV, confirming good colloidal stability.

A

B


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Figure 4 - TEM images of QDs. A) Hydrophobin encapsulated CdSeS/ZnS quantum dots, deposited from water (scale bar = 20 nm); B) Hydrophobin encapsulated CdSeS/ZnS quantum dots, deposited from water showing evidence of clustering on the grid (Scale bar = 50 nm). Hydrophobin-stabilised QDs (CdSe/ZnS) were subject to analytical ultracentrifugation in order to compare the conjugates to freely dissolved hydrophobins.18 We found that the distribution of sedimentation coefficients was bimodal with a very small shoulder/feature at the lowest sedimentation coefficients (Figure S2). This shoulder corresponded to about 0.01 mg/ml of non-adsorbed protein, as quantified by the refractive index detector during fractionation. The molar mass of this fraction (as derived from the distribution of sedimentation coefficients) ranged from 50 to 150 kDa and was hence comparable with free hydrophobin. Evaluating the main peak of the distribution of sedimentation coefficients, a diameter distribution with D50 = 4.2 nm, D90 = 9.7 nm was obtained. An independent experiment with a UV/VIS detector synchronized to the centrifuge, tuned to 520 nm, confirmed that this distribution was selective for the QDs and did not contain signal from any macromolecules. Since the hydrodynamic diameter values matched the diameters from TEM and dynamic light scattering data, we can conclude that the QDs are extremely well-dispersed in water without measurable agglomeration (

Hydrophobin-Encapsulated Quantum Dots - ACS Applied Materials

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