Article pubs.acs.org/Langmuir
Hybrid Assemblies of Fluorescent Nanocrystals and Membrane Proteins in Liposomes Vincenzo De Leo,†,‡ Lucia Catucci,†,‡ Andrea Falqui,§ Roberto Marotta,§ Marinella Striccoli,‡ Angela Agostiano,†,‡ Roberto Comparelli,*,‡ and Francesco Milano*,‡ †
Department of Chemistry, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy CNR-IPCF Istituto per i Processi Chimici e Fisici, Sez. Bari, Via Orabona 4, 70126 Bari, Italy § Nanochemistry, Istituto Italiano di Tecnologia (I.I.T.), Via Morego 30, 16163 Genova, Italy ‡
S Supporting Information *
ABSTRACT: Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo−NC−liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC− liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo−NC−liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.
1. INTRODUCTION Fluorescent semiconductor nanocrystals (NCs) are very attractive materials for a wide number of applications in different fields of science and technology. The strong and sizedependent tunable photoluminescence (PL) and the broad absorption spectrum, associated to a very high photostability with respect to conventional organic dyes,1,2 make them appealing for nanomedicine and biological applications.3,4 The related literature is being progressively enriched with applications involving in vivo and in vitro targeting, imaging, bioanalytical assays, drug delivery, disease treatment, and much more.5−8 On the other hand, the use of colloidal NCs for biorelated applications has been limited by difficulties in obtaining biocompatible nano-objects without affecting their peculiar features under aqueous biological conditions.9 In fact, the key goal to fully exploit NC properties is getting high control over their size, shape, and surface chemistry.10 Although several techniques have been proposed to directly synthesize NCs in water,11 they are not suitable for a wide range of NCs. Conversely, one of the most promising synthetic approaches relies on the thermal decomposition of suitable precursors in © 2014 American Chemical Society
the presence of terminating agents able to coordinate the NC surface to control the growth rate and prevent aggregation. Such an approach results in very stable NCs with high control over size and shape.12 The as-synthesized NCs are surrounded by a layer of terminating agents inferring stability in time against aggregation and making the inorganic NCs soluble in organic solvents. Nevertheless, the as-synthesized NCs are not water-dispersible and therefore show poor affinity to a biological environment.5 Various strategies have been developed to prevent such a drawback, based on the modification of the NC surface chemistry, preventing their aggregation, and retaining their peculiar optical properties: exchange of ligands on the surface, coating with silica shells, polymers, natural hydrophilic molecules, and incorporation in micelles.13−17 An alternative and very promising approach exploits the NC insertion into phospholipid-based liposomes.18 Received: October 28, 2013 Revised: January 23, 2014 Published: January 26, 2014 1599
dx.doi.org/10.1021/la404160b | Langmuir 2014, 30, 1599−1608
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Article
of CdSe@ZnS-loaded liposomes were checked by dynamic light scattering (DLS) measurements and cryo-EM investigation. The structural and functional survival of RC was verified by recording the visible-near infrared (vis-NIR) spectra and the charge recombination kinetics. This work creates new perspectives in both nanomedicine and nanotoxicity: hybrid liposomes can be used in nanomedicine for the realization of theranostic systems, i.e., with capability of transporting in cells both protein complexes with therapeutic activity and nanostructured agents useful for cell imaging and other diagnostic applications. Furthermore, they can be used in nanotoxicology to broaden the knowledge on the interaction between nanomaterials and integral membrane proteins, which today is limited for the most part to soluble proteins.
Liposomes are small vesicles that spontaneously form when lipid molecules are dispersed in an aqueous medium. Lipid molecules organize themselves to form a double-layer structure which is, in principle, identical to the natural cell membranes and that traps an aqueous volume in their core. Indeed liposomes mimic the lipid scaffolding of biological membranes and have well-characterized physicochemical properties and phase behavior.19 The population of formed vesicles can be tuned from tens of nanometers to tens of micrometers in diameter.20 Their appeal is derived from composition, which makes them biocompatible and biodegradable, and from wellestablished clinical use.21 Therefore, they arouse great interest from researchers as ideal carriers for biomedical imaging, drug delivery, targeted therapy, and biosensing.22 Several authors have described the incorporation of metallic,23 semiconductor,24 and amorphous25 nanostructured materials in liposomes, using various experimental strategies. Nanomaterials were either directly synthesized within liposomes26,27 or, more commonly, inserted after synthesis in a liposomal system.24,28 The fate of nanoparticles depends on their surface characteristics: hydrophilic ones can be easily trapped inside the aqueous compartment of the vesicles,29,30 while hydrophobic ones can be embedded within the lipid bilayer.31,32 The latter possibility is obviously more interesting: to use liposomes as carriers for transferring hydrophobic nanomaterials in an aqueous environment, changing their affinity phase, and, at the same time, providing a biocompatible shield against the biological environment.33 For this purpose, different approaches have been used such as incorporation into preformed liposomes of nanoparticles dispersed with detergent, bulk hydration of a dried film of lipids and nanoparticles, emulsion processes, and reverse phase evaporation methods.18,19,24,30,31,34−37 In this work, red-emitting hydrophobic CdSe@ZnS NCs were incorporated into liposomes of various phospholipid composition by the micelle-to-vesicle transition (MVT) method.38 This method offers a user-friendly strategy for the incorporation of hydrophobic NC into the bilayer of vesicles having narrow size distribution and, in addition, enables the simultaneous insertion of integral membrane proteins, resulting in the production of hybrid vesicles. The construction of liposomes simultaneously containing NCs and integral membrane proteins is described here for the first time by the successful coincorporation of CdSe@ZnS NCs and the photosynthetic reaction center (RC) from the bacterium Rhodobacter sphaeroides. The RC molecular weight is about 100 kDa, and the shape is approximately elliptical with axes of 3 and 7 nm. It represents a good model for studying the interaction between proteins and NCs in the phospholipid bilayer, because protocols for its purification and reconstitution in the membrane are well tested39 and the presence of bacteriochlorophylls as cofactors in its structure ensure strong and well-assigned signals through optical spectroscopic techniques.40,41 The effectiveness of the incorporation procedure was investigated as a function of NC surface chemistry (namely the chemical nature of the capping layer), NC concentration, type of detergent (nonionic, cationic, and anionic), and bilayer composition (zwitterionic, anionic, and poly(ethylene glycol) (PEG)-modified lipids were tested). The surface chemistry and the optical properties of the NCs were investigated by Fourier transform infrared (FT-IR), absorbance, and photoluminescence spectroscopy. The size, the morphology, and the stability
2. EXPERIMENTAL SECTION Materials. All chemicals were purchased with the highest purity available and were used without further purification. Cadmium oxide (CdO, powder 99.5%), selenium (Se, powder 99,99%), oleic acid (OLEA, technical grade 90%), trioctylphosphine oxide (TOPO, 99% and technical grade), tributylphosphine (TBP, 99%), trioctylphosphine (TOP, technical grade), tert-butylphosphonic acid (TBPA), diethylzinc (1.0 M solution in heptane), 1-octanethiol (OTT), 1-dodecanethiol (DDT), and hexamethyldisilathiane (HMDT) were purchased from Aldrich. Hexadecylamine (HDA) was purchased from Fluka. The reagent grade salts for the 50 mM K-phosphate, 100 mM KCl (pH 7.0) buffer solutions, sodium cholate (SC), octyl glucoside (OG), cetyltrimethylammonium bromide (CTAB), phosphatidylglycerol (PG), diphosphatidylglycerol (cardiolipin, CL), and phosphatidylserine (brain extract, type III: Folch fraction III from bovine brain, PS) were purchased from Sigma. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG2000-PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PLT) were purchased from Avanti Polar Lipids. Phosphatidylcholine (PC) was a kind gift from Lipoid. Synthesis of CdSe@ZnS Nanocrystals. CdSe@ZnS colloidal core−shell NCs were prepared by using a slightly modified literature synthetic procedure.42 All the synthetic steps were performed under standard airless conditions using a Schlenk line. In a typical synthesis, CdO (1 mmol) was dissolved in a TOPO:HDA:TBPA mixture (15:25:1). The resulting mixture was heated at 290 °C, and 2 mL of TBP was added to the reaction mixture. CdSe nucleation was promoted at 300 °C upon sudden injection of Se precursor solution, consisting of 5 mmol of Se dissolved in 20 mmol of TBP. The NC growth was carried out at 270 °C for 10 min. After that, the temperature was lowered to 110 °C and the NCs were annealed for 1 h to promote surface reconstruction before growing the ZnS shell. This step was performed using the same reaction mixture without any intermediate purification step, raising the temperature to 150 °C. The proper amount of a Zn(C2H5)2:HMDT (1:1) stock solution in TBP (50 mmol) was dropwise injected, and the shell growth was monitored by UV−vis absorption and PL spectra. The as-prepared core−shell NCs were precipitated by methanol and recovered by centrifugation three times. Then the obtained reddish powder was dissolved in CHCl3 for optical and structural characterization. CdSe@ZnS Capping Exchange. The capping exchange procedure exploits the strong affinity of thiol groups for the CdSe@ZnS NC surface. Here several alkyl thiols, basically differing in their alkyl chain length (i.e., in their steric hindrance), were tested, namely OTT, DDT, EDT, and PLT. In a typical procedure, alkyl thiol was added to a CHCl3 solution of washed CdSe@ZnS NCs (usually in molar ratio 2000:1) and vigorously stirred for 24 h at room temperature under ambient atmosphere. The excess of capping ligand was removed by repeatedly washing via the standard precipitation/centrifugation procedure. Then the precipitate was dissolved in the proper amount of CHCl3 for incorporation in liposomes. The effectiveness of the capping exchange procedure was assessed by UV−vis absorbance 1600
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the solution or suspension of interest (3−5 μL) on the upper face of the diamond crystal and allowing the solvent to evaporate. Dynamic Light Scattering. Dynamic light scattering measurements (DLS) were performed using a HORIBA Dynamic Light Scattering Particle Size Analyzer LB-550 instrument (Horiba Jobin Yvonne), equipped with a laser diode source (wavelength 650 nm, 5 mW). Measurements were performed at 25 °C with 100 sampling. The experimental time-course changes in the measured light intensity are converted in the frequency distribution, or Power Spectrum (PS), by fast Fourier transformation. The particle size distribution is then obtained by comparing iteratively, using the Twomey method, the experimental PS with calculated frequency distribution based on the intensity of the Brownian movement for particles of different size. Cryoelectron Microscopy. For cryo-EM analysis, 3 μL of each sample was deposited on Quantifoil grids (Electron Microscopy Sciences) previously glow-discharged in a Solarus plasma cleaner (Gatan, Inc.). Grids were automatically vitrified in a Vitrobot Mark IV (FEI) cryosample plunger. TEM images were recorded using low-dose conditions at −170 °C using a Titan Krios electron microscope (FEI) equipped with a FEG (field emission gun) and operating at 300 kV. Images were recorded using a FEI Falcon Direct Electron Detector.
measurements, steady-state and time-resolved photoluminescence spectroscopy, and FT-IR-ATR measurements. Encapsulation of the CdSe@ZnS Core−Shell NCs in the Phospholipid Bilayer of the Liposome. NC−liposomes were prepared by suitable modification of the micelle-to-vesicle transition (MVT) method, consisting of the detergent removal from phospholipid-containing mixed micelles to induce liposome formation.38 Several nonionic and anionic phospholipids were tested, such as PC, PG, CL, and PS, either separately or in mixture.43 For the sterically stabilized liposomes, PEG-2000-PE was added to the lipid blend. Lipids and NCs were dissolved in CHCl3 to give the desired ratio and then dried with a gentle nitrogen flux to form a homogeneous film on the walls of a conical glass tube. Solvent removal was completed under vacuum conditions (24 h) by a no-oil pump operating at 1 mbar. After that, 0.5 mL of 4% SC, or OG, or CTAB in 50 mM K-phosphate/100 mM KCl (pH 7.0) was added to the dry lipid film and then sonicated (20 shots with a Branson Sonicator 250) to form a clear, translucent mixed micelle solution. The latter was loaded into a glass column (20 × 1 cm) packed with G-50 Sephadex Superfine (Sigma) equilibrated with 50 mM K-phosphate/ 100 mM KCl (pH 7.0) for detergent removal by size exclusion chromatography (SEC). NC−liposomes eluted after a void volume of about 1.5 mL. Encapsulation of Photosynthetic RC in the Phospholipid Bilayer of the Liposome. RC was isolated from Rhodobacter sphaeroides strain R-26.1 as previously described.44 RC− and RC− NC−liposomes were prepared by the MVT method. The mixed micelle solution, obtained as described in the previous section, was added to 70 μM RC stock solution to a desired NC/RC ratio. The RC mixed micelle solution was vigorously shaken for 30 s and kept at 4 °C for 20 min before loading it on the column for the SEC. UV−Vis−NIR and Emission Spectroscopy. Absorption measurements were performed by means of a UV/vis/NIR Cary 5000 Spectrophotometer (Varian). The photoluminescence (PL) spectra were recorded by using an Eclypse Spectrofluorimeter (Varian). The relative PL quantum yields (QY) of NCs in solution and liposome suspension were estimated by using rhodamine 101 as reference dye and comparing the integrated PL intensity of the NCs and the dye, both recorded in excited solutions or suspension at the same absorbance (
