Polyelectrolyte

No dots can be found on their smooth surfaces, as indicated in Figure S3. Therefore, the visible small dots on composite ..... Finally, cell viability...
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In Situ Synthesis of Fluorescent Carbon Dots/ Polyelectrolyte Nanocomposite Microcapsules with Reduced Permeability and Ultrasound Sensitivity Hui Gao,† Andrei V. Sapelkin,‡ Magdalena M. Titirici,† and Gleb B. Sukhorukov*,† †

Materials Research Institute, School of Engineering and Materials Science, and ‡Centre for Condensed Matter and Materials Physics, School of Physics and Astronomy, Queen Mary University of London, London E1 4NS, United Kingdom S Supporting Information *

ABSTRACT: Designing and fabricating multifunctional nanocomposite microcapsules are considerable interests in both academic and industrial research aspects. This work first reports an innovative approach to in situ synthesize and assemble fluorescent carbon dots (CDs) into polyelectrolyte microcapsules, obtaining highly biocompatible nanocomposite microcapsules with excellent luminescence that facilitate imaging and identification in vitro, yet with the feasibility to load small molecules and ultrasound responsiveness to trigger their release. CDs are produced in situ in (PAH/PSS)4 microcapsule shells by carbonization of dextran molecules under relatively mild hydrothermal treatment. Compared with the collapsed and film-like (PAH/PSS)4 microcapsules, the novel composite microcapsules show a free-standing structure, smaller size, and thicker shell. CDs are proven to be fabricated and embedded in PAH/PSS multilayers, and the formed PAH/PSS/CD microcapsules are endowed with strong luminescence, as verified by the transmission electron microscopy, fluorescence spectra, and confocal laser scanning microscopy results. The in situ formation of CDs in capsule shells also empowers these capsules with ultrasound responsiveness and reduced permeability. The feasibility of encapsulation of small molecules (rhodamine B) and ultrasound-triggered release is also shown. Most importantly, due to the intrinsic biocompatible property and photostability of CDs, these fluorescent PAH/PSS/CD microcapsules show negligible cell toxicity and low photobleaching, which are impossible for capsules composited with conventional organic dyes and semiconductor quantum dots. KEYWORDS: carbon dots, in situ, fluorescent nanocomposite microcapsules, low permeability, ultrasound sensitivity

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including light and special ultrasound sensitivity to achieve triggered release. However, in this method, NPs need to be prefabricated and then assembled into PE multilayers, driven by weak forces such as electrostatic attraction, hydrogen bonding, and van der Waals interactions.12,13 A main drawback of this method is that the obtained composite capsules lack appreciable integrity and stability due to weak interactions between the NPs and polyelectrolytes (PEs). Besides, the inherent high permeability of LbL capsules cannot be reduced to allow encapsulation of small molecules (Mw ≤ 1000 Da).13−15 In situ growth of inorganic NPs in LbL structures

anoengineered organic/inorganic particles are being intensively studied for their ability to combine the advantageous properties of the constituent materials. A recent approach of interest to fabricate such heterostructures is embedding inorganic nanoparticles (NPs) within colloids, polymer micelles, or capsules, yielding multifunctional core/ shell composite particles.1−3 The formed hollow structures can serve as delivery vehicles, offering particular promise for applications in biomedicine.2 For example, it allows possible desired combination of fluorescence imaging, magnetic targeting, photothermal therapy, and drug delivery in one entity.3,4 Incorporation of magnetic NPs (Fe3O4),5,6 gold,7,8 and ZnO NPs1 within polyelectrolyte (PE) capsules has been achieved using a layer-by-layer (LbL) self-assembly route,9−11 empowering the composite capsules with novel properties © 2016 American Chemical Society

Received: July 29, 2016 Accepted: September 29, 2016 Published: September 29, 2016 9608

DOI: 10.1021/acsnano.6b05088 ACS Nano 2016, 10, 9608−9615

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Figure 1. (A) Schematic diagram of composite capsule fabrication, PAH (red lines), PSS (blue lines), and CDs (brown dots). (B) SEM and TEM images of capsules before (1−3) and after (4−6) CD incorporation.

is another way to incorporate various NPs and realize functionalization.16 Polymeric shells of capsules have roles as substrate or template, hence NPs can form, grow, and modify their morphologies, distributions, and assembly behaviors on such substrates according to the real situation, allowing the permeability to decrease and chemical stability of formed capsules to increase.17 Very recently, in situ growth of silica NPs in LbL capsules has been proven to be an effective method to reduce the shell permeability and enhance the stability of the formed composite capsules.13 Compared with the timeconsuming two-step procedures, growing NPs directly in target material opens a promising avenue to immobilize different NPs and engineer polymeric capsules. Here, we report a simple approach for one-step synthesis and assembly of fluorescent NPs to provide fluorescent capabilities and low permeability to the microcapsules that is based on conversion of dextran to luminescent carbon NPs. This approach has been inspired by recent numerous reports of

conversion of a variety of aromatic hydrocarbon to lightemitting carbon dots (CDs) under relatively mild conditions of hydrothermal carbonization18 and observation of efficient absorption of dextran into PE shells.19 Fluorescent CDs, which have been recently intensively explored, show excellent biocompatibility, low photobleaching, good chemical stability, and facile production with low cost.20−24 Many publications are related to the synthesis of CDs and their bioimaging applications.19,25,26 Recently, hollow CDs with ∼2 nm cavity size have been fabricated and used for both bioimaging and drug loading, but their loading capacity, limited by the small size, is extremely low.27 PE capsules, on the other hand, possess large cavity volume beneficial for drug delivery28,29 but have limitations due to their intrinsic high permeability and poor optical traceability.13,30 Poor traceability is usually addressed by incorporating fluorophores: tagging with fluorescent dyes and semiconducting quantum dots. This suffers from a number of problems intrinsic to these systems such as photobleaching, 9609

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Figure 2. (A) Fluorescence spectra of pure CDs (1) and PAH/PSS/CD capsules (2). (B) CLSM images of PAH/PSS/CD capsules with λex = 488 nm (left), λex = 543 nm (middle), and an overlay (right).

matrix, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied directly to characterize the capsules. Unlike the original flat, collapsed, and foldable (PAH/PSS)4 capsules (Figure 1B1−B3), the capsules after CD incorporation show a bowl-like shape with reduced size, thicker shells, and improved free-standing mechanics (Figure 1B4−B6 and Figure S2B,C). The inset TEM image confirms their hollow structure and indicates the shell thickness is ∼180 nm, which is around 10 times larger in comparison with the thin and transparent film-like PAH/PSS capsules (∼16−20 nm for 8 layers,31 Figure 1B3). The capsule size is statistically determined and decreases from 4.5 ± 0.37 to 1.56 ± 0.4 μm. To additionally probe the successful incorporation of in situ synthesized CDs, two control experiments were carried out. First, 2 mg·mL−1 dextran solution was hydrothermally treated under the same conditions without capsules, and the obtained yellow-brownish dispersion was analyzed by TEM. CDs with ∼5 nm size can be clearly seen from the TEM image (Figure S2A), similar to the ones observed as small dark dots on the surface of the composite capsule in Figure 1B6. Second, the PAH/PSS capsules autoclaved in pure H2O at 160 °C for 20 h are proven to shrink into filled balls with smooth surfaces, and the precipitation kept their original white color (Figure S3, Supporting Information). No dots can be found on their smooth surfaces, as indicated in Figure S3. Therefore, the visible small dots on composite capsule surfaces (Figure 1B6) belong to the slightly aggregated CDs. In terms of the structure change for (PAH/PSS)4 capsules under heating, the influence of hydrophobic forces greatly surpasses the electrostatic forces so that the shells rearrange their structure and shrink to decrease the surface area exposed to the solvent and finally appear as filled balls.30,32 In contrast, when treated at the same temperature, PAH/PSS/CD particles retain their hollow

toxicity, and poor stability in biological environments. Besides, there is a problem of additional complexity with dye integration into the microcapsule shells, as they need to be prelabeled with the employed polymers. To address these challenges and integrate the merits of CDs together with that of PE capsules, integrating CDs directly into PE shells is a promising approach, which could provide new opportunities for yielding multifunctional and traceable microcapsules. We report for the first time the in situ synthesis and assembly of fluorescent CDs in a PE microcapsule shell via a facile hydrothermal treatment.

RESULTS AND DISCUSSION The in situ synthesis and assembly of fluorescent CDs was achieved in poly(allylamine hydrochloride) (PAH)/poly(sodium 4-styrenesulfonate) (PSS) multilayers assisted by hydrothermal carbonization of dextran as a carbon source (Figure 1A). Initially, (PAH/PSS)4 capsules were obtained by LbL assembly technique elsewhere.13 Afterward, they were autoclaved in 2 mg·mL−1 dextran/H2O solution at 160 °C for 20 h. The resultant PAH/PSS/CD capsules were easily separated by centrifugation and purified from the solution by washing with DI water. Notably, the white color of the precipitation of capsules changes to yellow-brown after the hydrothermal reaction in the presence of dextran (Figure S1A, Supporting Information), as an indication that the carbon particles are synthesized. The Fourier transform infrared (FTIR) spectrum of the composite capsules indeed shows the characteristic CO (1723 cm−1) and C−O (1010 cm−1) stretching peaks of the pure CDs (Figure S1B, Supporting Information), which further indicates the formation of CDs from autoclaved dextran/H2O solution.18 To investigate the morphology and structure changes of the capsules and to validate the formation of CDs in the PAH/PSS 9610

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Figure 3. Characterization of reduced permeability. (A) CLSM images of PAH/PSS (1) and PAH/PSS/CD capsules (2) dispersed in RhB/ H2O solution (300 μg/mL). (B) CLSM images (1) and fluorescence spectra (2) of PAH/PSS/CD capsules with RhB encapsulated. The line scan insets show relative fluorescent intensity in capsules.

excitation-dependent emission (Figure 2A2) despite clearly similar PL spectral shape (Figure S5B, Supporting Information). This may be an indication that wavelength-dependent emission is an environment-related effect. It should be noted that PAH/PSS capsules hydrothermally heated in pure H2O exhibit no PL emission peaks. It supports the hypothesis that the fluorescence of PAH/PSS/CD capsules arises from the embedded in situ formed CDs via carbonization of dextran rather than any change/carbonization of the PAH/PSS multilayers. Fluorescent images of PAH/PSS/CD capsules taken using confocal laser scanning microscopy (CLSM) show numerous bright red and green circles (Figure 2B), which confirms that CDs are confined to the capsule shells. Furthermore, an overlay of images collected with λex = 488 nm and λex = 543 nm clearly shows that emission of different color/wavelength is localized to the same spatial regions of the polymeric shells. No significant changes in their fluorescent signal is detected after a continuous irradiation for 30 min with a 543 nm laser (Figure S6A, Supporting Information). Furthermore, their excellent fluorescent signal is maintained even after 8 months of storage in a refrigerator (5 °C) (Figure S6B, Supporting Information). These results indicate that the PAH/PSS/CD capsules are robust and stable and can be potentially applied for long-term imaging. It is worth mentioning that the same luminescent phenomena is also observed for a 2D PAH/PSS/CD membrane assembled on a glass substrate (Figure S7, Supporting Information), indicating that the approach is

structure despite the significant shrinkage. Reasonable explanations for such phenomenon include the following: (1) Dextran molecules attach to amino-terminated PAH and entangle with the PE multilayers, possibly facilitated by hydrogen bonds and hydrolyzable covalent cross-links resulting from aldehyde and primary amine coupling.19 (2) The in situ formed CDs buried within the multilayers (especially the positions with low energy, e.g., pores, concave surfaces, etc.) block the pores, bind together with PEs, reduce free space within the shells, and thus inhibit PE mobility while shrinking.17 (3) The entanglement of dextran and enforcement of in situ formed CDs in PE multilayers greatly prevent the shrinkage of PE caused by heat treatment. Besides, the formed CDs are negatively charged (−11.1 mV, Figure S4, Supporting Information), which potentially increases the electrostatic repulsion between the negatively charged layers and reduces the shrink effect caused by hydrophobic forces. Hence the interaction and combination of these factors are responsible for their hollow structure. Having established the morphology of CDs and their presence in capsule walls, we explored their optical properties. Absorption measurements (Figure S5A, Supporting Information) show the UV−visible (UV-vis) spectrum of CDs similar to those previously reported for CQDs/CDs.18,33 Furthermore, UV−vis excitation−emission spectra for CDs prepared from dextran show excitation wavelength (λex)-dependent emission similar to that observed in other CQDs/CD (Figure 2A1).18 Interestingly, photoluminescence (PL) emission of the CDs incorporated into the microcapsule shells does not show such 9611

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Figure 4. (A) Cell viability of the B50 cell line mixed with different concentration of PAH/PSS/CD capsules for 24, 48, and 72 h at 37 °C, as measured by an MTT assay compared with the control. The error bars show the standard deviations. The control is the cells without adding any type of capsules, and its viability is set as 100%. (B) CLSM image of PAH/PSS/CD capsules incubated with B50 cells for 6 h.

Figure 5. Characterization of ultrasound sensitivity. (A) SEM images of PAH/PSS/CD capsules after ultrasonication for (1) 0 s, (2) 2 s, (3) 10 s, (4) 20 s, (5) 60 s, and (6) 120 s. (B) Amount of released RhB from the composite capsules corresponding to the ultrasound treatment duration.

obviously not only limited to 3D structures but can also be explored as a general strategy for the accomplishment of luminescent 2D nanocomposite membranes. Subsequently, the PAH/PSS/CD capsule’s ability to encapsulate small molecules was investigated by employing a low Mw probe, rhodamine B (RhB, Mw = 489). RhB can

penetrate freely through PAH/PSS shells (Figure 3A1). The bright rings are due to RhB absorption within PAH/PSS shells. After the CDs’ in situ formation, the permeability for RhB drastically decreases (Figure 3A2), indicating a complete exclusion of RhB molecules from the composite shells (nearly zero fluorescence intensity inside capsules). As shown in Figure 9612

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ACS Nano 3B1, the capsules filled with RhB exhibit an average fluorescent signal intensity of ∼150 units inside and near zero unit outside the capsules. PL spectra in Figure 3B2 indicates that PAH/ PSS/CD capsules loaded with RhB exhibit emission peaks around 620 nm corresponding to RhB, meaning that RhB molecules are sealed inside the capsules. The above results prove a drastically reduced permeability of the formed capsules. Such phenomenon can be interpreted as a result of several factors, including shell shrinkage and densification induced by the heat treatment14,30 and the incorporation of the in situ formed CDs which could block pores and densify the shells.14 Toxicity, one critical issue during fabrication of the drug carrier, is a concern. No obvious cytotoxicity of CDs (concentration ≤200 μg·mL−1) to B50 cells is found by the measurement of MTT cell viability assays (Figure S8A, Supporting Information). The PAH/PSS/CD capsules (Figure 4A) show no significant changes in cell viability (all >80%), indicating an excellent biocompatibility. Compared with the MTT data of pure PAH/PSS capsules (Figure S8C) and the heated PAH/PSS capsules without CDs (Figure S8D), the PAH/PSS/CD capsules show a lower toxicity.34 The cell-tocapsule ratio shows slight variations in the toxic effect on B50 cells, exhibiting a slightly higher toxicity at the lower ratio (1:20) but still displaying cell viability of more than 80%. The cytotoxicity of PAH/PSS/CD capsules to B50 cells was also examined by the live/dead cell viability assay test result (Figure S8B, Supporting Information), which further proves their negligible toxicity. The uptake of PAH/PSS/CD capsules by B50 cells (cell-tocapsule ratio of 1:20, 6 h) was visualized by CLSM under laser irradiation at 543 nm. Figure 4B indicates that the microcapsules are internalized by B50, as evidenced by the fact that the capsules are localized within the same confocal plane as the enclosure of the cell membrane. Different from many soft LbL PE capsules, the internalized PAH/PSS/CD capsules retain their spherical morphology, showing only slight deformations (Figure 4B, inset) as a result of their mechanically strengthened thicker and denser shells. The statistic investigation on the uptake of the PAH/PSS/CD capsule in cells via CLSM reveals that ∼26.9% of the capsules in culture medium are taken up by B50 cells and ∼4 capsules are in one cell after 6 h of incubation, as shown in Figure S9 and Table S1. The CD uptake by B50 cells mainly exists in the cytoplasm and emits red fluorescence upon laser irradiation at 543 nm, while the nuclei are not luminescent (Figure S10, Supporting Information), demonstrating their potential usage as bioimaging probes. Ultrasound, which is already employed as a diagnostic and therapeutic method for many diseases, is a promising trigger due to its long penetration depth and low invasive nature.1 Here, we treated the PAH/PSS/CD capsules by ultrasound (GEX 750, Sonics & Materials, Inc., USA, 20 kHz, 50 W, 30% amplitude). SEM images of PAH/PSS/CD capsules after different sonication times are displayed in Figure 5A, demonstrating that the capsules are responsive to ultrasonication. The capsules show initially an intact bowl-like morphology, but some small fragments and broken capsules appear after only 2 s of ultrasonication, as indicated by the arrows in Figure 5A2. More capsules are broken into pieces after prolonged sonication time, and very few capsules are kept intact when time reaches 20 s. As seen from Figure 5A6, 120 s of sonication leads to complete destruction of the capsules, and only debris of the broken capsules is visible. An explanation for this enhanced ultrasound sensitivity possessed by the PAH/

PSS/CD capsules is in good agreement with previous reports regarding PE capsules functionalized with other inorganic NPs (e.g., ZnO, Fe3O4, SiO2).1,13,35 The inorganic NPs empower the formed shells with an increased stiffness and, therefore, increase the density gradient across the water/shell interface, consequently improving the absorption of acoustic energy.1,32 A quantitative measurement of the released RhB triggered by ultrasound was studied. To ensure that the release was not induced by diffusion, the release behavior of the microcapsules kept in the dark (to prevent photobleaching of RhB) without any treatment was monitored. It reveals that less than 4% of RhB is detected at t = 12 h and only ∼15% is released outside when the time is prolonged to 3 days (Figure S11, Supporting Information). Therefore, it can be concluded that RhB release due to diffusion is negligible within 300 s. Under ultrasonication, the amount of RhB increases rapidly within the first minute (Figure 5B). It rises to ∼17% after 6 s, ∼40% after 20 s, and sequentially reaches ∼50% after 60 s. The release curve becomes flat after 60 s, reaching a value of ∼80% at t = 4 min. No significant increase is detected with further increase of the treatment time to 5 min. It should be noted that not all of the encapsulated RhB is released (as detected), even though all capsules are ultrasonically broken due to the adsorption of RhB inside or on capsule shells.13 The reduced permeability and ultrasound sensitivity proven above indicate that the PAH/ PSS/CD capsules are promising for small molecule cages and triggered release by ultrasound. Additionally pure PAH/PSS capsules were heated in RhB/ H2O solution at 160 °C for 20 h and investigated. The CLSM image and the line scan inset in Figure S12A reveal that some RhB molecules are entrapped inside when they shrink into solid spheres. Upon ultrasonication the entrapped RhB molecules release slowly from the formed PAH/PSS spheres (Figure S12B). Only ∼3.4% of them release even after 120 s of ultrasound treatment as no broken spheres are found in the corresponding SEM images in Figure S13A−F, which is considerably different from the results of PAH/PSS/CD capsules upon ultrasound treatment. When the treatment time is prolonged to t ≥ 4 min, big fragments from PAH/PSS spheres appear (Figure S13G,H), but the amount of released RhB only reaches ∼3.9% at t = 5 min (Figure S12B), which means that most of the RhB molecules are still entrapped inside the broken pieces and only a small amount that located on the surface of the fragments is freed into the solution. The results indicate that it is a big challenge to release the encapsulated RhB from the PAH/PSS spheres. Hence, compared with PAH/ PSS/CD capsules, PAH/PSS spheres are not promising for delivery of small molecules. In comparison with previously reported microcapsules functionalized by prefabricated NPs, the composite microcapsules with in situ formed NPs offer many advantages, such as simplifying NP formation and incorporation process, excellent luminescent and mechanical stability, low permeability, and ultrasound sensitivity. These results confirm that in situ synthesis and assembly of CDs within PE microcapsules are a very elegant methodology to provide a nanoengineered platform that can combine fluorescent cell imaging, thermostable small molecule loading, and ultrasound-triggered release simultaneously.

CONCLUSIONS In summary, we have developed a one-step strategy for the synthesis and assembly of luminescent CDs as well as 9613

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centrifuged, and the supernatant was carefully collected, diluted, and used for RhB quantification. The fluorescence intensity of each sample (in supernatant) was determined and normalized with the standard fluorescent solutions with known concentrations. All of the samples were kept at the same dilution factors during the experiments, and the release amount was determined by the supernatant fluorescence intensity. For the RhB release from heated PAH/PSS spheres, PAH/ PSS capsules were heated in RhB/H2O solution (400 mg of RhB in 20 mL of H2O) at 160 °C for 20 h, and then they were washed, collected, and investigated in a similar way. Characterization. Scanning electron microscopy (FEI Inspect-F) was used to measure microcapsule morphologies. Diluted capsule suspension was dropped on glass slide, air-dried, and coated with gold. SEM observation was carried out using an accelerating voltage of 10 kV, a spot size of 3.5, and a working distance of approximately 10 mm. Transmission electron microscopy measurements were carried out using a JEOL 2010 transmission electron microscope with a LaB6 filament, operated at 200 kV. Samples were prepared by dropping the diluted microcapsule suspension on a copper grid with holey carbon film and letting it dry for 5 min. Infrared spectroscopy (FTIR spectrometer 100, PerkinElmer) was used to measure the FTIR spectra of vacuum-dried capsule samples, collecting data at a spectral resolution of 4 cm−1. UV−vis absorption spectra of CDs and dextran solutions were determined using a spectrophotometer (LAMBDA 950, PerkinElmer) and quartz spectrophotometer cuvettes (S10C, Sigma). CLSM images were obtained with a Leica TS confocal scanning system (Leica, Germany) equipped with a 63×/1.4 oil immersion objective. To test the permeability, equal volumes of capsule suspension and RhB (200 μg·mL−1) were mixed and shaken for 15 min before observation. Cell viability was assessed via a standard MTT assay and examined by a BMG Fluostar Galaxy plate reader. Fluorescence spectrometer (LS 55, PerkinElmer) was used to analyze fluorescence from the sample. Ultrasonic treatment was performed by an ultrasonic processor GEX 750 (Sonics & Materials, Inc., USA) operating at 20 kHz, 50 W, and 30% amplitude. The probe of the ultrasonicator inserted into a capsule suspension in a plastic tube. An ice bath was applied to ensure that the temperature change of the microcapsule suspensions was less than 5 °C.

entrapment of small molecules in PE multilayers assisted by a facile hydrothermal method. To the best of our knowledge, this is the first example of the polysaccharide-based in situ synthesis of fluorescent CDs embedding within PE multilayer shells. The as-prepared PAH/PSS/CD microcapsules exhibit excellent cytocompatibility, reduced permeability, good fluorescent stability, and ultrasound sensitivity, which provide an attractive alternative over the conventional dye-based capsule tracking and drug delivery vehicles. The integration of the fabrication and assembly procedures within one step provides a pervasive concept toward NP-functionalized PE microcapsules, and it could be potentially extended to synthesize libraries of various types of NPs to functionalize polymeric microcapsules and films.

EXPERIMENTAL SECTION Materials. Poly(allylamine hydrochloride) (PAH, Mw = 56 kDa), poly(4-styrenesulfonate) sodium salt (PSS, Mw = 70 kDa), dextran from Leuconostoc ssp (Mw = 10 kDa), rhodamine B (Mw = 479), CaCl2, Na2CO3, ethylenediaminetetraacetic acid (EDTA), and other chemicals were purchased from Sigma and used as received. Materials used for cell culture and cell viability studies including Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, trypsin, thiazolyl blue tetrazolium bromide (MTT), and methyl sulfoxide (DMSO) were also supplied by Sigma. Fabrication of Composite Microcapsules. CaCO3 cores were synthesized freshly by adding 0.33 M Na2CO3 solution into the same volume of 0.33 M CaCl2 while being vigorously stirred. Then alternating adsorption of polyelectrolytes (PSS and PAH, 2 mg·mL−1) onto the CaCO3 microparticles was carried out for 15 min with each layer followed by three Milli-Q washing/centrifugation steps (2500 rpm, 1 min). EDTA was used to remove the cores. The (PAH/PSS)4 microcapsules were then dispersed in dextran solution (2 mg·mL−1), heated at 160 °C for 20 h, and finally washed and collected for measurements. Cell Cultrure and Cell Viability Test. B50 rat neuronal cells were obtained from European Collection of Animal Cell Cultures (ECACC, Porton Down, UK). These cells are an ethylnitros urea-induced tumor cell line with neuronal morphology. They were cultured in the DMEM supplemented with 10% FBS and 1% penicillin−streptomycin containing 5% CO2/95% air at 37 °C. For cell viability studies, B50 cells were plated at 20 000 cells per well on 96-well plates. The next day, microcapsules were added at ratios of 5, 10, and 20 capsules per cell to triplicate wells. Similarly, CDs with different concentration dispersed in the cell culture medium were added to the wells. The experiment was terminated when total incubation times reached 24, 48, and 72 h for respective wells. Then 100 μL MTT solution (5 mg· mL−1 in cell culture medium) was added to each well, and plates were briefly shaken and then incubated for 3 h before DMSO was added. Finally, cell viability after coculturing with capsules for 24−72 h was assessed (compared with the control) and read by a BMG Fluostar Galaxy plate reader. Cellular uptake of capsules was visualized by a Leica SP5 and Leica TS confocal microscopes equipped with 63×/1.4 oil immersion objectives. RhB Encapsulation and Release Study. To examine the feasibility of small molecule encapsulation in formed composite microcapsules and further investigate their release behavior under ultrasound treatment, fluorescent small molecular dye, RhB (Mw = 479), was chosen as a model cargo substance. Generally, 400 mg of RhB was dispersed in 20 mL of dextran/H2O solution premixed with (PAH/PSS)4 capsules. Then they were heated at 160 °C for 20 h and finally washed to remove free fluorescent substances. The obtained RhB-containing capsule suspensions were split into two portions for further study: one was for the release study without any treatment, and the other was for ultrasound-triggered release. Then the suspensions were diluted into 20 mL. At specific time intervals, 1 mL of the capsule samples from these two portions with or without ultrasound treatment was taken out for measurement. The capsule cargo mixture was

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05088. Color change of microcapsule suspensions and precipitations before and after heating with dextran; FTIR profiles of CDs and microcapsules with and without CDs; TEM, UV−vis, and ζ-potential characterization of pure CDs; morphology and structure characterization (SEM and TEM) of PE capsules heated in H2O; photoluminescence emission data of PAH/PSS/CD microcapsules and that of CDs from dextran and glucose; photostability characterization of PAH/PSS/ CD capsules; CLSM images of PAH/PSS/CD films; cell viability data of CDs, pure PAH/PSS capsules, and heated PAH/PSS capsules without CDs; cell uptake images of PAH/PSS/CD capsules and their quantitative uptake data; cell uptake images of CDs; long-time release data of RhB from PAH/PSS/CD microcapsules; CLSM image of PAH/PSS spheres with RhB encapsulated inside; ultrasound-triggered release of RhB from the PAH/PSS spheres; SEM images of PAH/PSS spheres upon different ultrasonication time (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. 9614

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ACS Nano Notes

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The authors declare no competing financial interest.

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DOI: 10.1021/acsnano.6b05088 ACS Nano 2016, 10, 9608−9615