Magnetic-Nanoparticle-Decorated Polypyrrole Microvessels: Toward

Apr 18, 2013 - Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 ... Centre of New Technologies, University of Warsaw, Żwirki i Wigur...
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Magnetic-Nanoparticle-Decorated Polypyrrole Microvessels: Toward Encapsulation of mRNA Cap Analogues Krystyna Kijewska,† Anita Jarzębińska,† Joanna Kowalska,‡ Jacek Jemielity,‡,§ Daria Kępińska,† Jacek Szczytko,∥ Marcin Pisarek,⊥ Katarzyna Wiktorska,¶ Jarosław Stolarski,# Paweł Krysiński,† Andrzej Twardowski,∥ and Maciej Mazur*,† †

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Ż wirki i Wigury 93, 02-089 Warsaw, Poland § Centre of New Technologies, University of Warsaw, Ż wirki i Wigury 93, 02-089 Warsaw, Poland ∥ Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Hoża 69, 00-681 Warsaw, Poland ⊥ Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland ¶ National Medicines Institute, Chełmska 30/34, 00-725 Warsaw, Poland # Institute of Paleobiology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland ‡

S Supporting Information *

ABSTRACT: Many phosphorylated nucleoside derivatives have therapeutic potential, but their application is limited by problems with membrane permeability and with intracellular delivery. Here, we prepared polypyrrole microvessel structures modified with superparamagnetic nanoparticles for use as potential carriers of nucleotides. The microvessels were prepared via the photochemical polymerization of the monomer onto the surface of aqueous ferrofluidic droplets. A complementary physicochemical analysis revealed that a fraction of the nanoparticles was embedded in the microvessel walls, while the other nanoparticles were in the core of the vessel. SQUID (superconducting quantum interference device) measurements indicated that the incorporated nanoparticles retained their superparamagnetic properties; thus, the resulting nanoparticle-modified microvessels can be directed by an external magnetic field. As a result of these features, these microvessels may be useful as drug carriers in biomedical applications. To demonstrate the encapsulation of drug molecules, two labeled mRNA cap analogues, nucleotide-derived potential anticancer agents, were used. It was shown that the cap analogues are located in the aqueous core of the microvessels and can be released to the external solution by spontaneous permeation through the polymer walls. Mass spectrometry analysis confirmed that the cap analogues were preserved during encapsulation, storage, and release. This finding provides a foundation for the future development of anticancer therapies and for the delivery of nucleotide-based therapeutics.

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INTRODUCTION Nanometer-sized structures consisting of conjugated polymers have attracted considerable attention in recent years. A number of methods have been used to synthesize these materials; one of the most intensely studied methods is template synthesis.1−6 A template is defined as a structure that geometrically restricts the growth of a polymer, resulting in the formation of a polymeric replica. This method has been used to obtain spherical hollow structures, called capsules, or microvessels of polymers using colloidal particles as templates. The polymer is deposited onto a particle, and a thin layer is formed on the particle surface. The chemical or electrochemical oxidation of the corresponding monomer is typically used to produce the polymer layer on the colloidal particle.7−12 We have recently shown that photochemical polymerization can effectively be used to deposit a polymer onto liquid colloidal droplets.13−17 © 2013 American Chemical Society

One way to introduce new, unique functionalities to polymer structures is to incorporate solid nanoparticles (NP) into their walls. For example, polypyrrole microcapsules can be made susceptible to ultrasound-induced rupturing by incorporating gold nanoparticles into the microcapsules.13 This phenomenon is important from a medical applications point of view in regard to ultrasound-triggered drug delivery. Additionally, the incorporation of magnetic nanoparticles produces capsules with magnetic properties, enabling the vessels to be directed by an external magnetic field.16,18 The magnetic capsules can therefore be targeted to a therapeutic site in the body using an external field. Received: February 15, 2013 Revised: April 15, 2013 Published: April 18, 2013 1867

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Poland, reagent grade), HNO3 (POCh, Poland, reagent grade), KCl (POCh, Poland, reagent grade), tetramethylammonium hydroxide (TMAH, Sigma, reagent grade), KH2PO4 (POCh, Poland, reagent grade), Na2PO4 (POCh, Poland, reagent grade), NaCl (POCh, Poland, reagent grade), 5(6)-carboxyfluorescein (Sigma, 95%), N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, Aldrich, 97%), triethylamine (Sigma Aldrich, 99%), and DMSO (Sigma Aldrich, ACS reagent grade). Aqueous solutions were prepared using high purity water (Milli-Q Plus). Instrumentation. Scanning electron microscopy was performed using a LEO 435 VP (LEO Electron Microscopy Ltd., Cambridge, U.K.). Transmission electron microscopy was conducting using a Zeiss Libra 120 EFTEM or a JEM 1400 (JEOL Co., Japan, 2008). An energy-dispersive X-ray microanalysis system, Zeiss Merlin FE-SEM, was used to analyze the compositions of the capsules and to record maps of the elemental distributions. X-ray photoelectron (XPS) spectra were collected by a Microlab 350 instrument. The chemical compositions of the surface samples were characterized with AlKα, nonmonochromated radiation (1486.6 eV, 300 W) as the excitation source. During analysis, the pressure was 5.0 × 10−9 mbar. The binding energies of all of the analyzed elements were recorded for a pass energy of 40 eV with the binding energy of carbon (C 1s: 285 eV) as a reference. A linear or Shirley background subtraction was used to obtain the XPS signal intensity. The peaks were fitted with an asymmetric Gaussian/Lorentzian mixed function. X-ray microfluorescence measurements were conducted at the ID18 beamline of the European Synchrotron Radiation Facility (Grenoble, France). A monochromatic X-ray beam with an energy of 14.4 keV was used to record the spectra. X-ray powder diffraction was performed on the magnetic nanoparticles using a D8 Discover powder diffractometer (Bruker). Magnetic measurements were conducted using a SQUID magnetometer (Quantum Design MPMS XL-7T). A small, fresh, dry sample 0.45 mg in weight was placed in a Parafilm capsule and glued into a plastic straw. The sample was subjected to heating and cooling experiments over the temperature range of 2.0 to 300.0 K with a rate of 1 K min−1 at 0.01 T. ZFC/FC (zero-field cooling/field cooling) procedure was used to investigate irreversible or metastable state of the magnetic system. First the sample was cooled to 2.0 K without any magnetic field. Then a small external magnetic field of 100 Oe was applied, and the magnetization measurements were recorded during the heating up to 300.0 K. Finally the sample was cooled again with the same external magnetic field, and the magnetization was recorded. Hysteresis loops were measured at 2.0 K and at 300.0 K with the magnetic field from −7.0 to 7.0 T. Time-of-flight secondary ion mass spectrometry was conducted on a TOF.SIMS 5 (ION-TOF GmbH). The instrument was equipped with a liquid metal (Bi+) ion gun and with an O2+ source. Prior to analysis, the samples were sputtered with O2+ at 2 keV to remove any contaminates from the surface. For imaging and depth profiling in positive polarity, a Bi+ beam with an energy of 30 keV was scanned over a 40 μm × 40 μm area. Laser scanning confocal microscopy (LSCM) investigations were conducted using an Olympus IX70 FluoView 500 equipped with a 100x oil immersion objective. An argon ion laser (488 nm) was used as a light source. The fluorescence of fluorescein-tagged cap analogues was collected by a BP505 emission filter, and an image of the capsules in transmitted light was simultaneously recorded. The whole sample area was observed, and the most representative sample fragment was chosen for analysis. Fluorescence and steady state anisotropy data were collected by a Fluorolog FL3-2-IHR320 (Horiba-Jobin Yvon) spectrometer equipped with a 500 W xenon lamp. Ultraviolet−visible absorption spectra were recorded using a Lambda 25 (Perkin-Elmer) spectrometer. Thermogravimetric measurements were performed under an oxygen atmosphere using a TGA Q50 (TA Instruments). High-performance liquid chromatography (HPLC) was conducted using an Agilent Technologies 1200 Series instrument equipped with UV DAD and FLD detectors and with a SUPELCOSIL LC-18-T

The main advantage of polymer microvessels is that they contain a confined volume that can be loaded with guest molecules.8−13,16,17,19 Thus, these microvessels can be used, for example, as smart carriers for drug molecules. In the current paper, we demonstrate the preparation of magnetic microvessels loaded with mRNA cap analogues. A cap is a characteristic moiety present at the 5′ end of all eukaryotic messenger ribonucleic acids (mRNAs). The cap consists of a non-canonical nucleoside, 7-methylguanosine, which is linked to the first nucleotide of the mRNA chain by a 5′,5′-triphosphate bridge, and is often abbreviated m7Gppp(Np) n. The cap protects the mRNA from premature degradation at the 5′ end and is also involved in numerous steps in the mRNA life cycle. During the initiation of protein biosynthesis (mRNA translation), the mRNA cap is specifically recognized by the eukaryotic Initiation Factor 4E (eIF4E), which participates in the recruitment of ribosomes to the mRNA.20 eIF4E is involved in the translational control of gene expression and is therefore a protooncogene. Various types of tumor cells exhibit elevated eIF4E levels; these elevated levels have been linked to the augmented translation of the mRNAs, which are important in malignant transformation and metastasis.21 Targeting eIF4E is a way to inhibit the growth of cancer cells,22−24 which are more susceptible to cap-dependent translation than normal tissue.25 Numerous synthetic cap analogues have been shown to inhibit cap-dependent translation in vitro by competing with mRNA for the binding site of eIF4E.26−29 Therefore, the concept of using cap analogues in therapy as specific inhibitors to counteract the elevated eIF4E levels in tumor cells originated. However, due to the small size and presence of ionizable phosphate groups, cap analogues are unable to penetrate cellular membranes, similar to other small nucleotides. This problem is most likely one of the main reasons why the therapeutic potential of cap analogues has yet to be demonstrated in vivo. Usually, to overcome this problem, cap analogues are designed with a minimized phosphate backbone and with increased lipophilicity.30,31 This design, however, often leads to diminished biological activity because the triphosphate bridge significantly contributes to the stabilization of the eIF4E−cap complex.32 Another possible way to deliver nonpermeable drugs with therapeutic potential is to encapsulate them in liposomic or polymeric structures. One of the several materials used for this purpose is polypyrrole, which is a biocompatible material with relatively low cytotoxicity and immunogenicity.33−38 Moreover, the surfaces of polypyrrole particles can be modified with cell-targeting molecules, such as folic acid, to increase their uptake by cancer cells.39 Here, we report the preparation of polypyrrole microvessels modified with magnetic nanoparticles that can encapsulate cap analogues. We show that the nanoparticles retain their superparamagnetic state, leading to capsules with magnetic properties. The co-encapsulated cap analogues are located in the aqueous core of the microvessels and can be gradually released to the surrounding solution via permeation through the vessel walls.

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EXPERIMENTAL SECTION

Chemicals. All of the chemicals used in this study were of the highest quality commercially available: pyrrole (Aldrich, 98%), chloroform (Chempur, reagent grade), Fe(NO3)3·9H2O (Sigma, reagent grade), Ni(NO3)2·6H2O (POCh, Poland, reagent grade), Zn(NO3)2·6H2O (POCh, Poland, reagent grade), NaOH (POCh, 1868

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column 25 cm × 4.6 mm × 5 μm in size. Electrospray ionization mass spectrometry was performed using an AB Sciex API 3200. Synthetic Procedures. Synthesis of Ni0.5Zn0.5Fe2O4 Nanoparticles. Mixed nickel−zinc ferrite nanoparticles with the general formula Ni0.5Zn0.5Fe2O4 were synthesized in our laboratory, as described in our previous work.40,41 These ferrites were prepared using the “bottom-up” technique of co-precipitating nanoferrites from a solution of their precursors using a strong base. The synthesis was conducted by mixing a heated aqueous reaction mixture of the nitrate salts Fe(III), Ni(II), and Zn(II) in proper molar ratios with a hot aqueous solution of sodium hydroxide. It is crucial to maintain the reaction temperature above 80 °C because, at lower temperatures, the resultant hydroxides do not undergo dehydration and therefore do not form mixed ferrite oxides. Once the synthesis was terminated (after approximately 12 h of vigorous mixing at 90 °C under reflux), the brown suspension was allowed to cool to ambient temperature and magnetically precipitated using a strong permanent magnet. This precipitate was washed several times with cold deionized water. Finally, the slurry was converted into a ferrofluid by adding a few drops of tetramethylammonium hydroxide, yielding a clear sample with a deep ruby hue, pH 12. This ferrofluid was typically used to prepare nanoparticle-modified microvessels. For the co-encapsulation of nanoparticles and cap analogues, this ferrofluid was diluted by a PBS solution (phosphate buffered saline, 10 mM buffer, 140 mM NaCl, aqueous) to obtain a clear, brownish liquid with pH 6.4 prior to synthesis of the microvessels. This nearly neutral pH within the capsule core was required to prevent degradation of the cap analogues. Synthesis of Cap Analogues. Cap analogue 1 was synthesized as previously described.42 Cap analogue 2 was synthesized from a previously reported dinucleotide cap analogue with a 6-aminohexylene substituent at the N6-position of adenine, m7GpppANH‑(CH2)6‑NH2.43 The labeling of the cap with 5(6-carboxyfluorescein) was performed similarly to the method described by Jemielity et al. for labeling with biotin.44 Briefly, 5(6)-carboxyfluorescein was converted into its reactive NHS-ester by incubating it with TSTU and TEA in DMSO for 30 min, and the resultant solution was added portionwise over a period of 1 h to an aqueous solution of m7GpppANH‑(CH2)6‑NH2 buffered to pH 8.5. The product was purified using reversed-phase HPLC. Preparation of Polypyrrole Microvessels. Chloroform (5 mL) and water (25 μL) were mixed and sonicated for 30 s at 400 W using an ultrasonic homogenizer. Pyrrole (200 μL) was then added to the resulting emulsion. The mixture was poured into a cylindrical quartz container and rotated at 20 rpm for 4 min while being exposed to UV light from a mercury lamp (Polamp-5, Poland, 80 W), which was placed 10 cm from the reaction vessel. After polymerization was complete, the resulting microvessels were centrifuged at 13,400 rpm and washed with chloroform three times. To prepare solid-supported microvessels (for the TOF-SIMS experiments), the pyrrole-containing emulsion was poured into a Teflon container with a quartz window and irradiated by a mercury lamp 23 cm from the container for 6 min. This procedure resulted in the formation of hemispherical polymer structures supported on the quartz slide. The quartz window was then washed with chloroform and dried. Modification of Microcapsules with Magnetic Nanoparticles. The entrapment of magnetic nanoparticles was accomplished by using a ferrofluid instead of water to prepare the polypyrrole microcapsules, as described above. Loading the Microvessels with Cap Analogues. The encapsulation of cap analogues was similar to the incorporation of magnetic nanoparticles. An aqueous solution of 1 or 2 (1 mg/mL) was used to prepare the emulsion, followed by photopolymerization. To coincorporate the magnetic nanoparticles and cap analogues, an aqueous suspension of nanoparticles (12.5 μL, pH = 6.4) and a solution of a cap analogue (12.5 μL) were mixed and used to prepare the emulsion. Release of 2 from the Microvessels. A suspension of cap analogue 2-containing microvessels was dispersed in a pH = 7.3 buffered solution, 1 mg in 2 mL, and left overnight. The mixture was then centrifuged, and the supernatant was freeze-dried, redissolved in 50 μL of deionized water, and again centrifuged at 14,000 rpm. The resulting

supernatant was then analyzed by reversed-phase HPLC (0−50% gradient of MeOH in 0.05 M CH3COONH4, pH = 5.9 within 15 min). A peak eluting at 14.5 min, which was attributed to released 2, was measured and analyzed by an electrospray ionization mass spectrometer (AB Sciex API 3200).

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RESULTS AND DISCUSSION Polymer microvessels were prepared via the photochemical deposition of polypyrrole onto the surface of aqueous droplets dispersed in a chloroform phase. Pyrrole is known to undergo polymerization in chlorinated or brominated solvents when exposed to UV light. Although the exact mechanism of this process is not clear, it is believed that photoinduced electron transfer between the monomer and the solvent molecule occurs, yielding a pyrrole radical cation and a halogen anion. The pyrrole radical cations then couple to form a polymer, while the generated halogen anions become embedded in the polymer backbone, acting as counterions.13,17,45 A scanning electron microscopy (SEM) image of the polypyrrole microvessels prepared via the photopolymerization of the monomer onto aqueous droplets suspended in chloroform is shown in Figure 1a. Spherical particles with

Figure 1. (a) SEM image of the polypyrrole microvessels. (b) TEM image of an individual polypyrrole microvessel. (c) TEM image of a polypyrrole microvessel modified with magnetic nanoparticles.

average diameter of 900 nm can be observed (a histogram showing size distribution of the particles is shown in Figure S1, Supporting Information). The transmission electron microscopy (TEM) image of an individual microvessel confirms the presence of a hollow region within the particle (Figure 1b). The estimated vessel wall thickness is approximately 60 nm. Our next goal was to incorporate magnetic nanoparticles (MNPs) into the microvessels. For this purpose, we chose Zn0.5Ni0.5Fe2O4 particles because of their high saturation magnetization of ca. 73 emu/g. Moreover, with oxygen being present under biological conditions, both Zn2+ and Ni2+ cations 1869

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demonstrate that the nanoparticles constitute approximately 55% (w/w) of the microvessels modified with MNPs. An important question is whether the nanoparticles are incorporated into the microvessel polymer walls or if they are located within the hollow core. To answer this question, we recorded a series of TEM images of the capsule at various sample holder angles. These images were then used to make a video clip (Video1.avi, Supporting Information), which clearly shows that the nanoparticles are not located within the capsule core but are accumulated in or near the polymer walls. It appears that a fraction of the nanoparticles is directly embedded in the polymer walls, but the remaining species adhere to the inner side of the walls. This arrangement is likely due to the evaporation of the solvent from the capsule core. To confirm the identity of the nanoparticles contained in the microvessels, we used X-ray fluorescence spectroscopy (XRF). The XRF spectrum of the MNP-decorated capsules is shown in Figure 2b. Several spectral lines attributable to the elements in the nanoparticles are visible. The signals at 6.40 and 7.06 keV are assigned to the Kα1 and Kβ1 lines of Fe, respectively. Correspondingly, the Kα1 and Kβ1 signals of Ni are observed at 7.48 and 8.26 keV. The K lines of Zn are observed at 8.64 and 9.57 keV. These results clearly indicate that the dark spots observed in the TEM image are indeed Zn0.5Ni0.5Fe2O4 magnetic nanoparticles. While XRF probes the entire volume of the sample, providing the elemental composition, X-ray photoelectron spectroscopy (XPS), a related technique, examines only the surface of the sample. The XPS spectrum of the polymer microvessels decorated with magnetic nanoparticles is shown in Figure 3. Interestingly, no peaks attributable to iron, nickel, or

are resistant toward oxidation as compared with, e.g., magnetite (FeFe2O4), which is easily oxidized into maghemite (Fe2O3).46 To this end, we prepared a magnetic ferrofluid in an aqueous phase and used it to prepare the water-in-chloroform emulsion. After adding the monomer to the reaction mixture, the sample was exposed to UV light from a mercury lamp. This procedure yielded spherical particles, which were similar to those without MNPs. The corresponding TEM image (Figure 1c) shows a number of nanoparticles that are scattered within the polymer capsule (the size distribution histogram of the particles is shown in Figure S2, Supporting Information). To estimate the weight percentage of the incorporated nanoparticles, we used thermogravimetry. The microvessels decorated with MNPs were heated to 900 °C under an oxygen atmosphere to decompose the polymer phase, leaving the nanoparticles intact, and to determine the mass loss. Thermograms of polymer microvessels with and without MNPs are shown in Figure 2a. For the microvessels modified with MNPs,

Figure 3. XPS spectrum of the MNP-modified polypyrrole microvessels.

zinc are observed in the spectrum; these peaks should be exhibited at 707 eV (Fe2p3), 720 eV (Fe2p1), 853 eV (Ni2p3), 870 eV (Ni2p1), 1022 eV (Zn2p3), and 1045 eV (Zn2p1). This finding confirms that the magnetic nanoparticles are embedded within the polymer walls of the microvessel at a depth of at least several nanometers; thus, the nanoparticles are not detected by XPS. In the spectrum, signals at approximately 285 and 403 eV are observed, which are attributable to carbon and nitrogen, respectively.47 These elements originate from polypyrrole. The gold signals exhibited in the spectrum are due to the solid support. The origin of the oxygen peak at approximately 530 eV is unclear but is likely attributable to oxygen substituted into the polymer chain, e.g., in the form of carbonyl or hydroxyl groups.48 A small signal at approximately

Figure 2. (a) Thermograms of the polypyrrole microvessels: (i) modified with MNPs and (ii) not modified with MNPs. (b) XRF spectra of (i) the magnetic nanoparticles and (ii) the MNP-decorated microvessels.

the mass remains stable up to 200 °C and then gradually decreases, which is attributed to polymer decomposition. At approximately 500 °C, the mass stabilizes, reaching approximately 55% of the original mass. A reference sample of nonmodified capsules shows that these capsules are decomposed with no solid residue. The above data clearly 1870

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Figure 4. TOF-SIMS concentration profiles of selected ions for the silica-supported microvessels modified with MNPs: (a) C+, (b) Si+, (c) Fe+, and (d) 66Zn+.

Figure 5. TOF-SIMS images of the silica-supported microvessels modified with MNPs. Distribution images of (a) Fe+, (b) Ni+, and (c) Si+ ions after cumulative sputtering with an O2+ ion beam for 140 s. Reconstructed cross sections through the sample for (d) Fe+, (e) Ni+, and (f) Si+.

(TOF-SIMS). For this study, the microvessels were supported on a quartz plate. The aqueous droplets were first adsorbed onto the quartz slide, and the pyrrole was then photopolymerized onto their surfaces. A representative SEM image of the resulting structures is shown in Figure S4 (Supporting Information). The produced supported microvessels are hemispheres adjacent to the quartz slide. This shape makes the microvessels a convenient model system of nonsupported capsules for TOF-SIMS studies. To investigate the composition of the microvessels, the sample was sputtered with O2+ ions at 10 s intervals and successively analyzed using a Bi+ ion beam.

198 eV is due to chlorine. The analysis of the Cl2p core-level spectrum (Figure S3, Supporting Information) reveals that chlorine exists in two forms. The deconvolution of the Cl2p peak yields two components at 197.0 and 200.3 eV. The peak at the lower binding energy is assigned to a chloride anion incorporated into the polymer as a doping ion. The peak at 200.3 eV is due to chlorine covalently attached to the polymer chain.17 The incorporation of magnetic nanoparticles and their distribution within the polymer microvessels was further studied by time-of-flight secondary ion mass spectrometry 1871

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The concentration profiles of selected species ejected from the sample as a function of sputtering time are shown in Figure 4. The C+ ion counts attributable to organic material are initially high, but the counts gradually decrease, reaching a steady level after approximately 120 s. This result suggests that the polymer wall is first detected and then sputtered off, exposing some organics from the capsule core. The second type of analyzed ion is Si+, which is due to the silica support. These ions are initially absent in the mass spectrum, but their counts gradually increase after approximately 40 s, forming a plateau at approximately 120 s. This result clearly indicates that sputtering with O2+ ions removes the outer polymeric layer and exposes the silica support. To study the distribution of the magnetic nanoparticles incorporated into the microvessels, Fe+ and Zn+ ions were analyzed. The Fe+ concentration profile is shown in Figure 4c. Initially, for sputtering times from 10 to 40 s, the Fe+ concentration slightly increases, which is followed by a rapid increase. A plateau is exhibited in the curve after approximately 90 s. A similar profile is observed for Zn+ (Figure 4d). These data can be interpreted as follows: The initial nonzero counts for sputtering times up to 40 s are attributed to the MNPs embedded in the polypyrrole layer, and the rapid increase in the counts after 40 s is likely due to the nanoparticles that are accumulated beneath the polymer walls. The above conclusions are further supported by TOF-SIMS imaging. Distribution images of Fe+, Ni+, and Si+ ions after cumulative sputtering with an O2+ ion beam for 140 s are shown in Figure 5. For Fe+ and Ni+, bright islands are exhibited in the images, which correspond to the nanoparticle aggregates that remain after removal of the polymeric layer. For Si+ ions (Figure 5c), dark spots can be observed in a bright background. This result can easily be explained by assuming that the nanoparticle islands locally mask the silica slide; thus, these masked spots appear as dark circles on the image. The reconstructed cross sections through the sample (indicated by the solid lines) are depicted in Figures 5d−f (Fe+, Ni+, and Si+ distributions). At the top of the images, dark areas are exhibited that correspond to a sample volume with low amounts of Fe+, Ni+, and Si+ ions, which is attributed to a polymer layer with a relatively low amount of embedded nanoparticles. Below this layer (lower part of the images), bright regions are exhibited for Fe+ and Ni+, which are assigned to a nanoparticle aggregate. In the Si+ image (Figure 5f), this region appears black (low concentration of Si+), while the remaining area is bright, confirming the high concentration of Si+ ions. Here, the black region corresponds to the nanoparticle aggregate, while the bright part is attributable to the silica slide directly beneath the polymer adlayer. The next important question associated with MNP-decorated polymer microvessels is whether these microvessels exhibit magnetic properties. To answer this question, we performed SQUID magnetization measurements. The Zn0.5Ni0.5Fe2O4 nanoparticles themselves are superparamagnetic. Due to small size they are a single magnetic domain with one magnetic moment. However, at sufficiently high temperatures, the magnetic moment can randomly flip directions, resulting in the loss of the net magnetization in the absence of an external field. We were interested whether MNPs incorporated into the polymer microvessels retained their superparamagnetic properties. Magnetization loops in a low field range for MNPdecorated polymer microvessels at 2.0 and 300.0 K are shown in Figure 6a. For 300.0 K, the curve exhibits practically no

Figure 6. Magnetization of the MNP-decorated polymer microvessels: (a) magnetization loops over a low field range at 2.0 (open circles) and 300.0 K (solid circles). (b) ZFC/FC magnetization results: heating (lower branch) and cooling (upper branch) curves recorded in 0.010 T.

hysteresis, which is characteristic of a superparamagnetic state. However, at the low temperature, a considerable hysteresis is observed due to blocking of the magnetic moments. From the zero-field cooling (ZFC) and field cooling (FC) procedure (Figure 6b), we estimate that the blocking temperature is approximately 100 K. The above findings confirm that at room temperatures (down to 100 K) the encapsulated nanoparticles are in a superparamagnetic state, which makes the MNPmodified microvessels promising for biomedical applications. In the absence of an external field, the nanoparticle-decorated microvessels should not tend to aggregate because the magnetic moments of the individual MNPs will be randomly oriented. However, when an external field is applied, the alignment of the magnetic moments results in a net magnetization of the microvessels. Thus, the microvessels (when suspended in a solution) can migrate along the direction of the magnetic field. We demonstrate the migration of suspended MNP-modified microvessels in Video2.avi (Supporting Information). Our next goal was to verify whether polymer microvessels can be used as carriers for an organic guest material. For this study, we used two types of labeled mRNA cap analogues (Scheme 1). The Se-tagged cap 1 was loaded into the microvessels by simply using its aqueous solution to produce the water-in-chloroform emulsion. After the addition of the monomer, the mixture was exposed to UV light to photopolymerize pyrrole onto the surface of the liquid droplets. To 1872

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with MNPs at the same time. The corresponding EDS spectrum (Figure S5, Supporting Information) confirms the presence of Se, P, Fe, Ni, and Zn. For comparison, the Fe map of a microvessel co-loaded with 1 and with MNPs is shown in Figure 7b. Additionally, we studied the encapsulation of an mRNA cap analogue tagged with fluorescein 2 (Scheme 1). Labeling the cap with a fluorophore enables the use of fluorescence techniques in our studies. A laser scanning confocal microscopy (LSCM) image of polypyrrole microvessels loaded with 2, which were suspended in water, is shown in Figure 8a. Bright spots can be observed in the fluorescence image. The corresponding transmitted light image (Figure 8b) confirms that the chromophore is located in the capsules. To verify the identity of the encapsulated species, we recorded the excitation and emission spectra of the suspension (Figure 8c). An emission band is observed at 515 nm, and the excitation spectrum reveals an intense band at 475 nm and a small hump at approximately 315 nm. These bands are characteristic of fluorescein (the spike-like structure in the excitation band at 475 nm is due to scattering effects of the xenon lamp light of the spectrometer). We were also interested whether encapsulation affects the motional dynamics of the cap analogue in any measurable way. For this study, we performed steady-state anisotropy measurements. The excitation anisotropy spectra of 2 in free solution and encapsulated in the microvessels are shown in Figure 8d. For fluorescein, the S0 ← S1 emitting transition moment is nominally parallel to the S1 ← S0 absorption transition moment. The maximum anisotropy value for parallel absorption and emission transition moments is 0.4; this value is usually smaller due to rotational diffusion.49 Thus, the steadystate anisotropy R should be positive and less than 0.4. We measure R values of 0.022 and 0.014 for encapsulated and free 2, respectively. Because the anisotropy values are fairly comparable, we assume that the encapsulation does not significantly affect the rotational diffusion of the entrapped species. A next important question is whether the encapsulated cap analogues can be released into the external medium. As a result of the elevated concentration of the cap analogues within the capsule, it is expected that the analogues will be released gradually due to permeation through the vessel walls. We studied this process using fluorescence spectroscopy. For this purpose we prepared MNP-modified microvessels loaded with 2 (the pH of the ferrofluid was adjusted to 6.4 using a phosphate buffer to prevent possible degradation of the cap analogues under basic conditions). The vessels were placed in a buffered aqueous solution (pH = 7.3), and the fluorescence intensity was recorded at 515 nm. The release curve is shown in Figure 9a. The fluorescence initially increases and then reaches a plateau after approximately 40 min. The release of the encapsulated 2 is also confirmed by LSCM. Fluorescence and transmitted light microscopy images of the MNP-modified capsules after release of the cap analogues are shown in Figure S6 (Supporting Information). The fluorescence image clearly reveals that emission occurs from the external solution and that the emission is lower in the capsules. The identity of the released material was confirmed by mass spectrometry. The aqueous solutions were recovered after studying the release curves by first concentrating and then freeze-drying them, and the recovered materials were analyzed by reversed-phase HPLC with UV detection at 260 nm.

Scheme 1. Structures of Labeled mRNA Cap Analogues Used in This Study

detect the cap analogue, we used the electron microprobe mapping facility of the SEM microscope. The Se map of an individual polypyrrole capsule loaded with 1 is shown in Figure 7a. A round-shaped bright island can be observed in the dark background. The map indicates that the Se concentration is significantly higher inside the microvessel than outside the vessel, where the concentration remains at the noise level. Similarly, the microvessels can be loaded with both 1 and

Figure 7. (a) EDS Se map of an individual polypyrrole capsule loaded with 1. (b) EDS Fe map of an individual polypyrrole capsule co-loaded with 1 and with MNPs. 1873

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Figure 8. Polypyrrole microvessels loaded with 2 and suspended in water: (a) LSCM fluorescence image, (b) transmitted light image, (c) excitation and emission spectra, and (d) anisotropy excitation spectra (i, encapsulated 2; ii, 2 in free solution).

molar absorption coefficient 1.21 × 104 M−1 cm−1 at 465 nm (determined spectrophotometrically), and the absorbance value reveal that the amount of cap released is very close to the amount of cap used for preparation of the microvessels. Thus, the release efficiency is close to 100%.

A peak attributed to cap 2 (tR = 14.5 min, Figure 9b inset) was measured and analyzed using electrospray mass spectrometry. The recovered material was compared to a stock sample of 2. The ES(−) MS spectrum of the recovered material exhibited an m/z = 620.6 (Figure 9b), which corresponds to a doubly charged pseudomolecular ion. The fragmentation pattern observed in the MS/MS spectrum was comparable to an analogous spectrum of the control sample (Figure S7, Supporting Information). Hence, the cap analogue 2 is at least partially preserved during encapsulation, storage, and release. This result is important because nucleotides and cap derivatives in particular are chemically labile species, prone to hydrolytic degradation under various conditions.50 For preserving the intact cap structure proved to be the pH of the aqueous phase used for pyrrole polymerization. When the NPs used for encapsulation with cap 2 were suspended in an alkaline medium (pH ∼11), a biologically inactive, ringopened form of cap 2 was eventually released from the microvessels (m/z = 629.6 for a doubly charged ion; Figure S8, Supporting Information). However, when the NPs were buffered in a pH 6.4 medium, the cap remained intact. On the basis of the absorbance values, we were able to estimate the release efficiency of 2. The absorption spectrum of the solution after the release of 2 from the MNP-modified microvessels is shown in inset to Figure 9a. A rough calculation, assuming the

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CONCLUSION We have shown that the photochemical polymerization of pyrrole onto the surface of ferrofluidic droplets results in the formation of micrometer-sized capsule structures modified with magnetic nanoparticles. Our complementary physicochemical analysis reveals that the nanoparticles are embedded in the microvessel walls and in the capsule cores. The incorporated nanoparticles exhibit superparamagnetic properties; thus, the MNP-modified microvessels can be directed by an external magnetic field. Additionally, the magnetic capsules can be loaded with nucleotide-derived guest molecules. We have demonstrated the encapsulation of mRNA cap analogues, which have been recently proposed as agents for cancer therapy. We confirmed that the cap structure is preserved during encapsulation and subsequent release. This finding paves the way for studying polypyrrole capsules as carrier vehicles for nucleotides with therapeutic potential. The simplicity of the preparation, the variety of techniques available for analyzing the cap analogue-loaded microvessels, and the 1874

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Figure 9. (a) Release curve of 2 from the microvessels modified with MNPs: fluorescence intensity at 515 nm versus time with inset showing the UV−vis spectrum of released 2. (b) ESI MS(−) spectrum of released 2 with inset showing the reversed-phase HPLC profile of released 2.

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previously confirmed therapeutic potential of cap analogues make the cap analogue loaded microvessels prepared in this study model objects for future biological studies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

ASSOCIATED CONTENT

The authors declare no competing financial interest.

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S Supporting Information *

ACKNOWLEDGMENTS This work was supported by the National Science Centre, project nos. 2011/01/D/ST5/05869 and 2011/03/N/ST4/ 00750. We thank Dr. Birgit Hagenhoff and Dr. Reinhard Kersting for performing the TOF-SIMS experiments. The measurements were performed at the ION-TOF GmbH headquarters (Münster, Germany). Special thanks are owed to Dr. Rafał Bartosiewicz (Nencki Institute of Experimental Biology) for performing the TEM measurements and to Dr. Marianna Gniadek (Department of Chemistry, University of Warsaw) for the EDS analysis. We also thank Dr. Jakub Jaroszewicz (Warsaw University of Technology) for his assistance with XRF and Paweł Majewski (Department of Chemistry, University of Warsaw) for the experimental work conducted on the preparation of the magnetic nanoparticles. The XRF measurements were performed at the European

Histograms showing the distribution of diameters of polymer microvessels and magnetic nanoparticles; XPS Cl2p core level spectrum of polymer microvessels modified with MNPs; SEM image of solid-supported microvessels; EDS spectrum of a polypyrrole capsule co-loaded with Se-tagged cap analogue and MNPs; confocal microscopy images of microvessels modified with MNPs after release of fluorescein-labeled cap analogue; MS characterization of the compound released from microvessels loaded with fluorescein-labeled cap analogue at alkaline pH; a video clip constructed from a series of TEM images of the MNP-modified capsule recorded at various sample holder angles; a video clip demonstrating migration of MNP-modified microvessels in external magnetic field. This material is available free of charge via the Internet at http://pubs.acs.org. 1875

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Synchrotron Radiation Facility (Grenoble, France) at beamline ID18 under project no. EC-725. The TEM equipment was installed under the project sponsored by the EU Structural Funds: Centre of Advanced Technology BIM−Equipment purchase for the Laboratory of Biological and Medical Imaging. The EDS measurements were conducted using the research equipment purchased for the CePT project, which was cofinanced by the European Union from the European Regional Development Fund under the Operational Programme Innovative Economy 2007−2013. Steady-state fluorescence measurements were conducted at the Structural Research Laboratory (SRL) at the Department of Chemistry, University of Warsaw, Poland. The SRL was established with the financial support of the European Regional Development Fund under the Sectoral Operational Programme ″Improvement of the Competitiveness of Enterprises, years 2004-2006″, project no. WKP_1/1.4.3./1/2004/72/72/165/2005/U. The HPLC and MS measurements were performed in the Biopolymers Laboratory, Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, supported by the ERDF within the Innovation Economy Operational Programme POIG.02.01.00-14-122/09

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