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Clickable, Biocompatible, and Fluorescent Hybrid Nanoparticles for Intracellular Delivery and Optical Imaging Markus Mu¨llner,† Anja Schallon,‡ Andreas Walther,†,§ Ruth Freitag,‡ and Axel H. E. Mu¨ller*,† Makromolekulare Chemie II and Bayreuther Zentrum fu¨r Kolloide und Grenzfla¨chen, Universita¨t Bayreuth, 95440 Bayreuth, Germany, Bioprozesstechnik, Universita¨t Bayreuth, 95440 Bayreuth, Germany, and Molecular Materials, Department of Applied Physics, Helsinki University of Technology, FIN-02015 TKK, Finland Received September 28, 2009; Revised Manuscript Received November 20, 2009
We report a general and facile approach for the fabrication of a new class of near monodisperse hybrid nanoparticles via RAFT polymerization and self-assembly in water. Furthermore, we combine a fluorescent inorganic silica core with a biocompatible polymer shell and a terminal unit susceptible to facile conjugations via click chemistry. A tailoring of the weight fractions of both components allows a tuning of the size of the formed aggregates. Fluorescent properties and the crosslinking into an organic-inorganic hybrid network are realized by copolymerizing a dye-functionalized monomer 1-pyrenebutyl acrylate and a trimethoxysilane-carrying one, (3-acryloxypropyl)trimethoxysilane. The potential of these stabilized and fluorescent nanoparticles as biocompatible carriers for intracellular delivery is demonstrated via in vitro experiments on lung cancer cells.
Introduction The development of biocompatible nanoparticles for molecular imaging and targeted therapy is of considerable current interest.1-5 Fluorescent nanoparticles have immense potential in a number of biotechnological applications, such as biological imaging or sensor technology.6-8 These applications require size-controlled, monodisperse, bright nanoparticles that can be specifically conjugated into biological macromolecules. Nanoparticles with a core-shell architecture have the extra benefit of providing a robust platform for incorporating diverse functionalities into a single nanoparticle.9,10 Unfavorable solubility, stability, and toxicity (e.g., CdSe) all hinder the therapeutic efficacy of many probes for optical imaging and can thereby prevent the approval of investigational probes for clinical use.11 Drug delivery systems12-17 help to provide a concentration of drugs in an aqueous milieu well above the solubility limits of the free drug by targeting delivery to the required cells. The method can be used furthermore to deliver organic dyes;18 for example, pyrene into cells for imaging purposes.19 Features like tunable light emission20 and superior signal brightness give outstanding possibilities for new routes in cellular imaging and bioconjugation. Fluorescent nanoparticles with targeting ligands, like antibodies, peptides, or small molecules, can be used for targeting tumors with high affinity and specificity. Quantum dots (QD) are also of special interest for biological applications.4,21,22 However, they are still limited in use due to their toxicity, uneasy functionalization, and limited biocompatibility. Furthermore, water solubility is required for medical and most biosensing applications. An elegant alternative to quantum dots was introduced by Wiesner and co-workers.23 They developed a synthetic strategy to produce fluorescent core-shell silica nanoparticles. The * To whom correspondence should be addressed. E-mail: axel.mueller@ uni-bayreuth.de. † Makromolekulare Chemie II and Bayreuther Zentrum fu¨r Kolloide und Grenzfla¨chen, Universita¨t Bayreuth. ‡ Bioprozesstechnik, Universita¨t Bayreuth. § Helsinki University of Technology.
nanoscale fluorescent material exhibits covalently bound organic dyes surrounded by a robust silica shell. The so-called CornellDots (C dots) offer very high levels of emission brightness and an absence of blinking, typical of quantum dots. Various methods of delivering nanoparticles within the body have been investigated. One actively studied system for delivery purposes is block copolymer micelles. These are spherical, nanosized, supramolecular assemblies of amphiphilic block copolymers that possess a core-shell-type structure. The core of the micelles is the cargo area that accommodates predominantly hydrophobic substances, and the shell is a brushlike protective corona that ensures the water dispersibility of the micelles. The advantage of polymer particles as carriers of drugs, in contrast to administration of free drugs, is the increased circulation time in the body.24 The reticuloendothelial system (RES) can detect such polymer particles and eliminate them from blood circulation. Surface alteration of the particle, however, can delay or prevent recognition by the RES. Poly(ethylene glycol) is known to substantially enhance the circulation time due to its excellent protein repellent properties.25 Clearance of drug-loaded particles was also found to be considerably lower for particles less than 200 nm, which was assigned to a higher surface curvature.26 The so-called enhanced permeability and retention (EPR) effect leads to the preferred accumulation of polymers in the tumor, while the ineffective lymph drainage of tumors hampers the clearance of the drug carrier. Despite the high thermodynamic and kinetic stability of polymeric micelles, further stabilization is often necessary to avoid disintegration of the aggregate at low concentrations or upon environmental changes, such as elevated temperature, altered pH values, or increased ionic strength. An easy and promising approach to achieving a more robust delivery system consists of crosslinking the micelles into stabilized nanoparticles.9,27-29 The incorporation of hydrophobic and often toxic agents into water-soluble, biocompatible, block copolymer micelles can lead to targeted delivery at the subcellular level. In this study, we combined several attractive concepts for the simple and fast preparation of biomedical carriers. In detail,
10.1021/bm901099p 2010 American Chemical Society Published on Web 12/21/2009
Hybrid Nanoparticles for Intracellular Delivery
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Scheme 1. Preparation of Clickable Hybrid Nanoparticles via Self-Assembly and Crosslinking in Water
we exploit the controlled self-assembly of a block copolymer with two designed functionalities into well-defined nanoscopic colloids. The core-forming block can be easily stabilized toward a silica-like nanoparticle. This core is rendered fluorescent via the copolymerization of dye molecules. The hydrophilic corona is protein repellent and enables a straightforward click conjugation29-31 at its periphery, which can in principle be used for receptor targeting. We chose RAFT polymerization for the preparation of the block copolymers, as it provides some of the best means to polymerize a wide variety of monomers with good control over the end group functionality. The amphiphilic block copolymers are composed of a hydrophilic poly[oligo(ethylene glycol) acrylate] POEGA segment and a second hydrophobic block containing trimethoxy-functionalized and dye-substituted monomers. The hydrophobic block can be crosslinked into a silsesquioxane network, which has the dye molecules entrapped within the inorganic-organic hybrid network. The polymerization is mediated by an alkyne-carrying chain transfer agent (CTA), enabling copper-catalyzed alkyne-azide click chemistry at the periphery (Scheme 1). After studying the formation of the colloidal aggregates and their crosslinking, the nanoscopic carriers were tested with respect to their biocompatibility and cellular internalization on lung cancer cells. The accessibility of the click function was then demonstrated by the conjugation to another fluorescent dye.
Experimental Section Materials. Benzene (anhydrous), 1-pyrenebutanol (99%), 2-propyn1-ol (propargyl alcohol, 99%), triethylamine (TEA, >99%), 4-dimethylamino pyridine (DMAP, 99%), N,N′-dicyclohexylcarbodiimide (DCC, 99%), potassium ferricynide (K3Fe(CN)4, 99%), rhodamine B (dye content ∼95%), 3-chloro-1-propanol (98%), and sodium azide (>99%) were purchased from Sigma-Aldrich and used without further purification. Acryloyl chloride (97%), benzylmagnesium bromide solution (1 M in THF), and carbon disulfide (99%) were purchased from Fluka and used as received. Dichloromethane (DCM), methanol, benzene, and dioxane were used in p.a. grade. N,N-Azobisisobutyronitrile (AIBN) was ordered from Aldrich and recrystallized from ethanol. Oligo(ethylene glycol) acrylate (OEGA) with number-average weight Mn ) 454 g/mol was purchased from Aldrich and freed from inhibitor by passing through a basic alumina column. (3-Acryloxypropyl)trimethoxysilane (APTS, 95%) was purchased from ABCR and freed from inhibitor by passing through a silica column. Polymerizations. Synthesis of ω-Alkyne-poly[oligo(ethylene glycol) acrylate] (PEOGA-CTA). A mixture of propargyl (4-cyanopentanoic acid)-4-dithiobenzoate) (0.25 g, 0.8 mmol), oligo(ethylene glycol) acrylate (OEGA, 107 g, 240 mmol), and anhydrous benzene (100 mL) in a screw-cap flask, sealed with a rubber septum, was deoxygenated
by bubbling with nitrogen. In a second flask, a stock solution of AIBN (30 mg, 0.18 mmol) in anhydrous benzene (10 mL) was deoxygenated similarly. Afterward, 8.8 mL of this stock solution were transferred to the reaction mixture with a syringe and the polymerization flask was placed into an oil bath at 65 °C. Samples were withdrawn to monitor conversion. The polymer was purified via dialysis in dioxane. Freezedrying yielded the final pure marco-CTA. Synthesis of ω-Alkyne-poly[oligo(ethylene glycol) acrylate]-b-poly[(3acryloxypropyl)-trimethoxysilane]-co-poly(1-pyrenebutyl acrylate). Alkynefunctionalized poly[oligo(ethylene glycol) acrylate]-CTA precursor (4.5 g, 0.092 mmol, Mn ) 48.000 g/mol) was dried in a screw-cap flask under vacuum at 40 °C overnight, then dissolved in 22 mL anhydrous benzene. To the mixture, (3-acryloxypropyl) trimethoxysilane (8.7 g, 37 mmol) and 1-pyrenebutyl acrylate (3.02 g, 9.2 mmol) were added. The solution was degassed by bubbling with nitrogen. In a second flask, a stock solution of AIBN (10 mg, 0.06 mmol) in anhydrous benzene (5 mL) was degassed similarly. Afterward, 1.5 mL of this stock solution was transferred to the reaction mixture with a syringe and the polymerization flask was placed into an oil bath at 70 °C. To obtain polymers with different block lengths, various samples at different times of conversion were withdrawn. All obtained polymers were dialyzed for several days in benzene before freeze-drying was performed. The synthesis of the chain transfer agent (Figure 1) was conducted similarly as reported earlier by Goldmann et al.32 The synthesis of azidorhodamine B was accomplished via esterification of rhodamine B with an azide function-carrying alcohol. Characterization. SEC. SEC in THF was conducted at an elution rate of 1 mL/min using PSS SDVgel columns (300 × 8 mm, 5 µm): 105, 104, 103, and 102 Å and RI and UV (λ ) 254 nm) detection. A poly(tert-butyl acrylate) calibration curve was used to calibrate the columns, and toluene was used as an internal standard. NMR. 1H and 13C NMR spectra were recorded on a Bruker AC-250 spectrometer in various solvents at room temperature. DLS. Dynamic light scattering (DLS) measurements were carried out at a scattering angle of 90° on an ALV DLS/SLS-SP 5022F compact goniometer system with an ALV 5000/E cross-correlator and a He-Ne laser (λ0 ) 632.8 nm). The CONTIN algorithm was applied to analyze the obtained autocorrelation functions. Cryo-TEM. For cryogenic transmission electron microscopy (cyroTEM) studies, a drop of the sample dissolved in water was put on a lacey transmission electron microscopy (TEM) grid, where most of the liquid was removed with blotting paper, leaving a thin film stretched over the lace. The specimens were instantly vitrified by rapid immersion into liquid ethane and cooled to approximately 90 K by liquid nitrogen
Figure 1. Chemical structure of the used chain transfer agent.
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in a temperature-controlled freezing unit (Zeiss Cryobox, Zeiss NTS GmbH, Overkochen, Germany). The temperature was monitored and kept constant in the chamber during all of the sample preparation steps. After freezing the specimen, the specimen was inserted into a cryotransfer holder (CT3500, Gatan, Mu¨nchen, Germany) and transferred to a Zeiss EM922 EF-TEM instrument. Examinations were carried out at temperatures around 90 K. The transmission electron microscope was operated at an acceleration voltage of 200 kV. All images were registered digitally via a bottom-mounted CCD camera system (Ultrascan 1000, Gatan) combined and processed with a digital imaging processing system (Gatan Digital Micrograph 3.9 for GMS 1.4). Formation of Aggregates and crosslinking. Micelles were prepared by dissolving the polymers (c ) 0.1 g/mL) in dioxane at room temperature. Subsequently, the polymer solution in dioxane was dialyzed into methanol and then water. The crosslinking of the poly[(3-acryloxypropyl)trimethoxysilane] (PAPTS) domains was performed in water at 50 °C. A total of 30 mL of polymer solution (c ) 0.1 g/mL) was stirred vigorously, while a total amount of 0.5 mL of aqueous ammonia solution (1.6 vol%) was added within 2 days. Dialysis of the crosslinked polymer aggregates in Milli-Q water removed the residual aqueous ammonia. Click Reaction. A total of 0.71 g POEGA107-(PAPTS71-PPBA20) (1.0 × 10-5 mol) was brought into a 250 mL two-neck round-bottom flask. DMF (150 mL) was added and was degassed using two freeze-pump-thaw cycles. Afterward, 0.08 g (0.5 × 10-3 mol) CuBr and 0.225 g (1.0 × 10-3 mol) 2,2′-bipyridyl (bipy) were introduced and connected to an addition funnel containing 8 mg azido-rhodamine B dissolved in 30 mL of degassed DMF. Once the addition to the rhodamine solution was completed (40 min), the reaction was allowed to proceed at 80 °C for 2 h. After cooling to ambient temperature, the product was purified by exhaustive dialysis. Cell Culture. A549 epithelial cells were purchased from LGC Standards ATCC (Wesel, Germany) and were cultured in DMEM (PAA, high glucose) supplemented with 10% FCS, streptomycin (100 µg/mL), penicillin (100 IU/mL), and 2 mM L-Glutamine. Cells were cultivated at 37 °C in a humidified 5% CO2 atmosphere. MTT Test. Cytotoxicity of polymers was determined in 96-well microtiter plates using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) conversion assay. A549 cells (carcinomic human alveolar basal epithelial cells) were seeded at a density of 1 × 104 cells/well 24 h prior to each experiment. Before each assay, the culture media was replaced by a media without serum and antibiotics to prevent interaction with the polymers. Cells were incubated for 24 h with different concentrations of polymer at 37 °C in a humidified 5% CO2 atmosphere. After incubation, cells were rinsed with PBS and further incubated in 200 µL of MTT solution (0.5 mg/ mL in phosphate buffered saline) for 2 h at 37 °C. The solution was aspirated and the formazan product was solubilized with dimethylsulfoxide (DMSO). Absorbance was measured at 580 nm in a microplate reader (Genios Pro, Tecan GmbH, Crailsheim). Untreated cells were used as controls. Microscopy. A549 cells were inoculated at a density of 2 × 105 cells/well in six-well plates on the day before. The culture media was replaced by media without FCS and antibiotics 1 h before the polymers were added to the cells. After 24 h of incubation, the cells were washed with PBS, fixed with 4% para-formaldehyde solution for 15 min and washed again with PBS. The fixed cells were counter-stained using 1 µg/mL nuclear dye (SYTOXGREEN) for 15 min. The cells were washed again with PBS and once with Milli-Q water. The coverslips were mounted in Prolong Gold mounting medium (Invitrogen) according to the manufacturer’s instructions. The cells were observed with an epifluorescence microscope (Olympus BX51TF, Hamburg, Germany). In addition, images were obtained using a laser scanning confocal microscope (Leica TCS-SP5, Leica, Wetzlar, Germany) equipped with argon, neon, and UV lasers. Fluorescence images were captured and processed with a digital imaging processing system (Leica TCS software, Leica, Wetzlar, Germany). The different excitation/
Mu¨llner et al. emission conditions for pyrene and SYTOXGREEN were used in separate channels with the X63 oil immersion objective. An image cutting horizontally through the cell was selected out of a z-stack of images.
Results and Discussion Synthesis and Molecular Characterization of Block Copolymers. Oligo(ethylene glycol) acrylate (OEGA) was chosen for polymerization of the first block because of its excellent protein repellence. Its good solubility in most common organic solvents and water guarantees good stabilization of the later aggregates in both organic solvents and water. It also allows for further reactions in a variety of solvents. The polymerization kinetic for OEGA using the clickable chain transfer agent (click-CTA) is shown in Figure 2. After an induction period of about 30 min, an almost linear increase in molecular weight with conversion can be observed, indicating a well-controlled RAFT process taking place. The polydispersity indices remain very low (PDI < 1.13) within the investigated conversion range. The SEC elution trace does not show any indications of termination via chain-chain coupling. (3-Acryloxypropyl)trimethoxysilane (APTS) was chosen for polymerization of the second block due to its ability to undergo crosslinking into a stable, inorganic silsesquioxane network under basic conditions. Additionally, 1-pyrenebutyl acrylate (PBA) was added as a comonomer (molar ratio APTS:PBA ) 4:1) to introduce fluorescent tags into the hydrophobic block. Furthermore, PBA can be considered a hydrophobic drug mimic. The kinetics of the copolymerization of APTS and PBA mediated with the POEGA macro-CTA show similar characteristics of a controlled polymerization. The polydispersity indices are very low (PDI < 1.16) at lower conversions, however, they increase to 1.23 and 1.42 at high conversions. The reason for the increase of PDI at high conversion is not due to termination reactions, but due to unreacted POEGA macro-CTA. The inactive POEGA-CTA precursor is most likely caused by some unwanted termination reaction, which occurs during the polymerization of the first block or from the decay of the CTA end group through exposure to light and to oxygen during storage. The SEC elution traces show no indication of termination reactions, such as coupling (Figure 2). We quenched the polymerization at rather low conversion (
