Microcapsules Containing pH-Responsive, Fluorescent Polymer

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Microcapsules Containing pH-Responsive, Fluorescent Polymer-Integrated MoS: Effective Platform for in-situ pH Sensing and Photothermal Heating 2

Chan Ho Park, Sangmin Lee, Ghasidit Pornnoppadol, Yoon Sung Nam, Shin-Hyun Kim, and Bumjoon J. Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19468 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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ACS Applied Materials & Interfaces

Microcapsules

Containing

pH-Responsive,

Fluorescent Polymer-Integrated MoS2: Effective Platform for in-situ pH Sensing and Photothermal Heating

Chan Ho Park1†, Sang Min Lee1†, Ghasidit Pornnoppadol 2, Yoon Sung Nam2, Shin-Hyun Kim*,1, and Bumjoon J. Kim*,1 1

Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea 2 Department of Material Science and Engineering, KAIST, Daejeon 34141, Republic of Korea

KEYWORDS: pH-responsive polymer, MoS2, microcapsules, FRET, photothermal therapy

1

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ABSTRACT We report the design of a novel microcapsule platform for in-situ pH sensing and photothermal heating, involving the encapsulation of pH-responsive polymer-coated MoS2 nanosheets (NSs) in microcapsules with an aqueous core and a semipermeable polymeric shell. The MoS2 NSs were functionalized with pH-responsive polymers having fluorescent groups at the distal end to provide pH-sensitive Förster resonance energy transfer (FRET) effect. The pH-responsive polymers were carefully designed to produce a dramatic change in polymer conformation, which translated to a change in FRET efficiency near pH 7.0 in response to subtle pH changes, enabling the detection of cancer cells. The pH-sensitive MoS2 NSs were microfluidically encapsulated within semipermeable membranes to yield microcapsules with uniform size and composition. The microcapsules retained the MoS2 NSs without leakage, while allowing the diffusion of small ions and water through the membrane. At the same time, the membranes excluded adhesive proteins and lipids in the surrounding media, protecting the encapsulated MoS2 NSs from deactivation and enabling in-situ pH monitoring. Moreover, the encapsulated MoS2 NSs showed high-performance photothermal heating, rendering the dual-functional microcapsules highly suitable for cancer diagnosis and treatment.

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1. INTRODUCTION Detecting pH changes near neutral pH is of crucial importance for clinical and biomedical applications. For example, cancer cells render their microenvironment weakly acidic (pH 6-7) through secretion of lactic acid, and therefore pH can be an effective indicator of tumor sites.13

However, it is difficult to measure the local pH of cellular environments by conventional

methods (e.g., pH meters or Litmus papers) due to limited accessibility of cellular environments in vivo and the large sample volume required for analysis. To overcome these limitations, pH-responsive polymers have been utilized as building blocks of gels4-8 and polymer shell-coated nanoparticles9, 10 for the optical sensing of pH in subcellular scale due to their precisely tunable pH-responsive behavior and high biocompatibility.11-14 In addition, a fluorescent molecule and optical quencher can be linked by pH-responsive polymers to yield complexes with pH-dependent Förster resonance energy transfer (FRET).15-18 As each complex reports the local pH through fluorescence intensity, the spatial distribution of pH can be directly visualized by fluorescence imaging. However, there remain many critical problems to achieve practical pH measurements in complex environments. First, it is difficult to synthesize pHresponsive polymers with high selectivity and reversibility near neutral pH condition because the transition pH of polymers often varies with their concentration and functional groups,19, 20 and the insoluble-to-soluble transition is often very slow.21, 22 Secondly, the polymers are usually deactivated when they are directly exposed to physiological fluids, as adhesive proteins and lipids can irreversibly distort or aggregate the polymers.23-25 In addition, dilution of the polymers reduces the fluorescence intensity and thus the signal-to-noise ratio, resulting in inaccurate measurements.26, 27 Therefore, materials capable of probing local pH with high sensitivity around neutral pH in a complex medium remain highly demanding yet unmet need. 3



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Two-dimensional nanosheets (NSs) of molybdenum disulfide (MoS2) have served as an efficient quencher for FRET due to their high absorbance over a wide range of wavelengths.28-31 The MoS2 NSs have a high surface area-to-mass ratio and numerous sulfur binding sites, enabling simple surface functionalization with oligomeric or polymeric molecules under mild conditions.32, 33 The high density of negative charges on the surface of MoS2 NSs allows for their stable dispersion in aqueous media,34 which can prevent the excessive aggregation when the NSs are functionalized with pH-responsive polymers. Moreover, the NSs possess a high extinction coefficient in the near-infrared (NIR) region, suggesting their use as efficient photothermal (PT) agents for the biomedical applications.35-38 Therefore, MoS2 NSs functionalized with pH-responsive polymers and fluorescent dyes can potentially serve as FRET-active materials for effective local pH measurement and PT heating for detection of cancer cells and simultaneous PT treatment, which, to the best of our knowledge, has not yet been reported. In this work, we designed FRET-based microsensors by attaching pH-responsive polymers containing fluorescent end-groups on the surface of MoS2 NSs (F-MoS2 NSs) and encapsulated them into microcapsules. The pH-responsive polymer linkers were random copolymers of 2-(diethylamino)ethyl methacrylate (DEA) and butyl methacrylate (BMA). Optimization of the ratio of the two monomers produced a conformational change in the pH range of 6.4 – 7.2, thereby yielding a dramatic change in fluorescence intensity as a function of pH around neutral pH through modulation of FRET. The F-MoS2 NSs were microfluidically enclosed by semipermeable membranes fabricated using a water-in-oil-in-water (W/O/W) double-emulsion template. The membranes allowed the diffusion of small ions while excluding large molecules, which precluded access of proteins such that the pH-responsive behavior of the MoS2 NSs was maintained in cell culture media. Moreover, the microcapsules prevented 4


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dilution of the encapsulated MoS2 NSs, providing a consistent fluorescence intensity upon deployment in cellular environments. The high concentration of encapsulated MoS2 NSs also benefitted localized PT heating, wherein the NS-loaded microcapsules increased the temperature by more than 30˚C upon NIR irradiation. These materials allowed temporal pH monitoring in a culture medium containing cancer cells and, upon NIR exposure, resulted in cell death.

2. EXPERIMENTAL SECTION 2.1 Materials. MoS2 powder (< 2 μm, 99%), n-butyllithium (1.6 M in hexane), potassium phosphate

monobasic

(KH2PO4),

potassium

phosphate

dibasic

(K2HPO4),

N,N'-

dicyclohexylcarbodiimide (DCC, 99%), 4-(dimethylamino)pyridine (DMAP, 99%) and other materials were purchased from Sigma-Aldrich. Sodium hydroxide (NaOH powder, 98%) and hydrochloric acid (HCl, 1 M) were purchased from Daejung Chemical & Metal Co. Azobisisobutyronitrile (AIBN, 98%) was purchased from Junsei Chemical Co. and purified by recrystallization from ethanol. Deionized water (18 MΩ·cm) was used in all experiments. The phosphate buffers used in these experiments consisted of a mixture of KH2PO4 and K2HPO4. By varying the amount of each salt, buffers with various pH conditions were prepared in the pH range between 5.8 and 8.0. Various sodium phosphate buffers of the desired pH (pH 6.0 – 8.0, interval = 0.2) were produced by adjusting the mixing ratio of 1 M KH2PO4 to 1 M K2HPO4 stock solutions. The pH was calculated according to the Henderson-Hasselbalch equation: pH = pK ′ + log⁡(

𝑝𝑟𝑜𝑡𝑜𝑛⁡ 𝑎𝑐𝑐𝑒𝑝𝑡𝑜𝑟 𝑝𝑟𝑜𝑡𝑜𝑛⁡ 𝑑𝑜𝑛𝑜𝑟

), where pK’ = 6.86 at 25°C.

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2.2 Characterization. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AMX 500 spectrometer. Transmission electron microscopy (TEM) (JEOL 2000FX) was performed to analyze the morphology and dimensions of the MoS2 sheets and the composites. TEM samples were prepared by dropping aqueous suspensions onto LC200-Cu grids coated with a holey carbon film, followed by drying in the oven (~70 oC). For scanning electron microscopy (SEM) measurements, the samples were dissolved in water and drop casted onto O2 plasma-treated Si wafers. Attenuated total-reflectance Fourier transform infrared (ATR-FTIR) spectra were acquired on a Bruker ALPHA spectrometer. Thermogravimetric analysis (TGA) was performed on a TA Q500 instrument at a scan rate of 10 oC min−1 from room temperature to 700 oC under a nitrogen atmosphere (99.999%). The photoluminescence (PL) spectra were obtained using a 10 mm quartz cuvette and a Horiba Jobin Yvon NanoLog spectrophotometer. Both PL and confocal microscopy were excited at 543 nm. For time-resolved fluorescence (TRF) measurement, the concentration of F-MoS2 was maintained to have an absorbance at 455 nm smaller than 0.1 to avoid optical quenching by other MoS2 substrates in the solution. Photographs were acquired with a digital camera. IR images were recorded with an FLIR 62101-0301 T440 high-sensitivity infrared thermal imaging camera (temperature range: -20 to 1200 oC). NIR exposures were performed using an 808 nm IR diode laser system (LVI808CW4WF-VA, LVI technology) and the laser power measured by a power meter (3Sigma, PM30). The formation of W/O/W double emulsions was observed using optical microscope equipped with a high-speed camera (MotionScope M3, IDT). The microcapsules were imaged using an upright microscope (Ni-U, Nikon) and a laser scanning confocal microscope (LSM 5 PASCAL, Carl Zeiss).

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2.3 Synthesis of Thiol-terminated Poly(DEA-r-BMA)-RB. DEA and BMA monomers were dissolved in tetrahydrofuran (THF), and the 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid chain transfer agent (1) and AIBN (0.05 mol% of (1)) were added to the solution in a glass ampoule (Figure S1). The reaction mixture was degassed by 3 times of freeze–pump-thaw cycles, and then heated at 70 °C. After 12 h, the reaction mixture was poured into a mixture of water and methanol to precipitate the copolymers, which were then centrifuged several times. After drying the sediment under vacuum, poly(DEA-r-BMA) (2) was obtained as a translucent pellet. The carbonothioylthio (S=C—S) end groups of poly(DEA-r-BMA) were converted to thiol groups (3) by reaction with excess hexylamine for 24 h to enable conjugation to the MoS2 NS. To synthesize poly(DEA-r-BMA)-RB, the carboxyl group of rhodamine B (RB) was first reduced to a hydroxyl group (RB-OH) (4) by following a previously reported protocol.39 Subsequently, poly(DEA-r-BMA) (0.08 mmol), RB-OH (0.16 mmol), and DMAP (0.02 mmol) were mixed in THF or dimethylformamide for 30 min. DCC (0.08 mmol) was then added to the mixture. After 36 h, the mixture was filtered through a 0.22 μm microporous membrane and then the solution was poured into water (P20 and 30 only) and hexane (P0-30) to precipitate the polymers. Subsequently, the precipitates were transferred to toluene and centrifuged (13000 rpm, 20 min) to remove organic residues. Finally, the supernatant was collected and dried (5).

2.4 Synthesis of the F-MoS2 NSs. MoS2 NSs were prepared from MoS2 powders according to previously reported procedures.35 MoS2 powder (300 mg) was immersed in stirred in a solution of n-butyllithium (3 mL, 1.6 M in hexane) for 48 h, yielding water-soluble single or fewlayered MoS2 NSs after purification. To prepare F-MoS2, an excess amount of thiol-terminated poly(DEA-r-BMA)-RB (40 mg) was added to a solution of MoS2 NSs in methanol (20 ml, 1 7



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mg mL-1). After sonication for 1 h and stirring for 12 h, residual polymers were removed by centrifugation several times at 25 oC, and removal of unattached polymer chains was confirmed using thin-layer chromatography. The obtained F-MoS2 were redispersed in desired pH buffer solutions.

2.5 Preparation and Operation of Microfluidic Device. The microfluidic device consisted of two tapered cylindrical glass capillaries coaxially aligned in a square glass capillary. One of the cylindrical capillaries was tapered by capillary puller and sanded to have 120 μm orifice, which was then treated with octadecyltrimethoxysilane (Sigma-Aldrich) to render the surface hydrophobic. The other tapered capillary was sanded to have 240 μm orifice, which was treated with 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (Gelest, Inc.) to render the surface hydrophilic. To form the innermost droplets of double emulsions, an aqueous dispersion of FMoS2 (0.53 g L-1 in pH 6.0 buffer solution) was injected into the hydrophobic capillary. The middle oil phase containing polysiloxanes modified with methacrylate (SB4722, Evonik) and 2 w/w% photoinitiator (Darocur 1173) was injected through the interstices between the hydrophobic capillary and square capillary. The continuous phase, a 10 w/w% aqueous solution of PVA (Mw 13000-23000 g mol-1, Sigma-Aldrich), was injected through the interstices between the hydrophilic capillary and square capillary. The volumetric flow rates of the innermost, middle, and continuous phases were set to 500, 135, and 3000 μl h-1, respectably, using syringe pumps (Legato 100, KD Scientific). The double-emulsion drops formed in the device were collected in distilled water, which was then irradiated with UV light for 30 s. The resulting microcapsules were washed with distilled water several times. The concentration of microcapsules was determined by counting the number of microcapsules in 10 μl drop of 8


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microcapsule containing stock solution using optical microscopy (OM). The evenly suspended stock solution contained approximately 365±4.3⁡ units of microcapsules in 10 μl (averaged from three trials), which corresponds to 36500 units ml-1. Using this stock solution, we prepared a series of the solutions containing different concentrations of capsules (2900, 4400 and 7300 units ml-1).

2.6 PT Heating Using an NIR Laser. A laser beam was used to irradiate a quartz cuvette containing the samples. The temperature was measured using a thermocouple at the edge of the cuvette to avoid blocking the laser beam and a digital laser gun thermometer. The quartz cuvette was covered with a cap to prevent the evaporation of water except when measuring the temperature. All solutions were equilibrated at room temperature before laser irradiation.

2.7 In vitro PT Therapy of A549 Cells. A549 cells (1×104) were seeded on a 96-well plate in RPMI medium and incubated for 24 h at 37°C. After the incubation, the microcapsules were added into the medium to yield number concentration of 7300 units ml-1 and further incubated for 3 days. The microcapsules were then irradiated with an NIR laser (808 nm, 1 W cm-2) for different times. After the irradiation, the cell viabilities were estimated using a CCK-8 assay and a microplate reader (λ=450 nm). In a duplicate test, histochemical staining was performed using trypan blue (0.4%).

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3. RESULTS AND DISCUSSION 3.1 Synthesis and Characterization of F-MoS2 NSs

Figure 1. (a) Schematic illustration showing the construction of the F-MoS2 NSs and pHdependent FRET, which is mediated by a conformation change of poly(DEA-r-BMA). (b) PL spectra of F-MoS2 NSs at three different pH values of 7.4, 7.0, and 6.0. Insets are corresponding confocal microscopy images of the solution. Scale bars are 100 μm. (c) pH-dependent PL intensity of the F-MoS2 NSs at the concentration of 0.53 g L-1 (excitation at 543 nm). For comparison, the dye solution was also tested, and showed no pH dependence. (d) Size distributions of pristine MoS2 NSs at pH 7.4 and F-MoS2 NSs at pH 7.4, 7.0, and 6.0, as measured by DLS.

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To produce a FRET-active pH-sensing material, we decorated the surfaces of MoS2 NSs with pH-responsive polymers containing a fluorescent end-group, RB (Figure 1a). The conformational change of the polymer linkers as a function of pH produced the changes in the distance between the RB dye and MoS2, which modulated the FRET efficiency and therefore the fluorescence intensity. To induce the conformation change under mildly acidic conditions, we

selected

poly[(2-(diethylamino)ethyl

methacrylate)-random-(butyl

methacrylate)]

(poly(DEA-r-BMA)) as the pH-responsive polymer linker. Poly(DEA) containing tertiary amines undergoes a transition between a deprotonated, hydrophobic state and a protonated, hydrophilic state around pH 7.4. Incorporation of the hydrophobic BMA moiety is known to decrease the transition pH of poly(DEA-r-BMA) copolymers.40, 41 Therefore, it was expected that the transition pH could be adjusted to mildly acidic conditions through optimization of the composition. To determine the optimum ratio of DEA to BMA, we synthesized a systematic series of poly(DEA-r-BMA) copolymers with five different [DEA]:[BMA] ratios and the RB dye was attached at one end of the polymer by esterification, as shown in Figure S1 of the Supporting Information. For the systematic control of the pH response, the number-average molecular weights (Mn) of poly(DEA-r-BMA)-RB were maintained to have similar values (15 – 25 kg/mol) (Table S1). The BMA fraction in the polymers was determined quantitatively by the proton NMR (1H NMR) spectroscopy to vary from 4 to 28 wt% in good agreement with the trend of Fourier transform IR spectra (Figure S2) and monomer feed ratios employed during the synthesis. End-functionalization of the polymers with RB was estimated to be >75% by comparing the values of the molar extinction coefficient of RB and poly(DEA-r-BMA)-RB (Figure S3). Post-polymerization aminolysis with hexylamine afforded thiol chain ends to facilitate tethering of the polymers to MoS2 NSs.

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We investigated the solubility of the poly(DEA-r-BMA)-RB copolymers in aqueous solutions with various pH values, ranging from 5.8 to 8.0 to evaluate the pH dependence of molecular conformation (Figure S4).20 The pH value at which the soluble-to-insoluble transition occurred was dependent on the fraction of BMA in the polymer chain: copolymers with higher fractions of BMA exhibited the transitions at lower pH, as expected due to the intrinsic hydrophobicity of the BMA units. The copolymer containing 20 wt% BMA (P20) exhibited a soluble-to-insoluble transition around pH 7.0, whereas P10 and P30 underwent similar transitions at pH 7.2 and 6.6, respectively. Therefore, P20 was selected for further studies to its pH sensitivity in the range between the physiological (pH 7.4-7.0) and cancer microenvironments (pH 6.0-6.8).1-3 The MoS2 NSs were prepared by chemical exfoliation with butyl lithium in aqueous solution according to previously reported procedures.34-36 The pristine MoS2 NSs showed hexagonal symmetry, as confirmed by TEM images and corresponding fast Fourier transform (FFT) patterns, indicating that the exfoliated MoS2 NSs remained intact (Figures S5a, b).42 The poly(DEA-r-BMA)-RB polymers were attached on the surface of the MoS2 NSs by a ‘‘grafting-to’’ method,43-45 involving the formation of a bond between the thiol end group of P20 with the surface of the MoS2 NSs.33 The successful anchoring of the polymers onto the FMoS2 NSs was supported with TEM images (Figures S5c-f); the curved boundaries were observed along the NSs only in the samples of F-MoS2 NSs, which were suspected to be the polymers. In addition, elemental analysis and their mapping acquired by energy dispersive spectroscopy showed the presence of nitrogen only for the F-MoS2 NS samples, indicating the presence of poly(DEA-r-BMA)-RB coatings (Figures S6). The conjugation between the poly(DEA-r-BMA)-RB and the MoS2 NSs was further supported by UV-Vis-NIR spectra and ATR-FTIR spectroscopies (Figure S7). The ATR-FTIR spectrum of the F-MoS2 NSs showed 12


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the stretched C-H and C=O stretching peaks at 2963 and 1727 cm−1, respectively, from poly(DEA-r-BMA)-RB, whereas that of the pristine MoS2 NS showed the broad peak around 3400 cm−1.46, 47 We also confirmed the anchoring of the polymers on the F-MoS2 NSs by thermogravimetric analysis, from which the areal chain density of the polymers on the surface of the MoS2 NSs was calculated to be 0.07 chain nm−2 (Figure S8).35, 48 The anchoring of the polymers on the MoS2 NSs significantly altered the dispersion properties (Figure S9). The pristine MoS2 NSs were partially dispersible in alcohols and formed large agglomerates in dichloromethane (DCM) and toluene.49 In contrast, the polymer-grafted MoS2 NSs were dispersible in alcohols, DCM, and toluene, consistent with the solubility of the poly(DEA-rBMA) copolymers in all of these solvents.

3.2 pH-dependent FRET Effect of F-MoS2 NSs We next examined the fluorescence intensity of the F-MoS2 NSs at different pH values, especially in the range of 7.4 - 6.0. The extracellular environment of the cancer cells has a mildly acidic pH, typically 6.8 - 6.0 due to the production of lactic acid by anaerobic glycolysis, whereas that of normal cells maintains a pH of 7.4 - 7.0.1-3 As P20 exhibits a conformational change at pH 7.0, the FRET efficiency is expected to change dramatically above and below pH 7.0, as illustrated schematically in Figure 1. PL spectra of F-MoS2 NSs show a peak at 577 nm, regardless of pH, from RB. However, the intensity at 577 nm significantly increased as the pH decreased from 7.4 to 6.0, as shown in Figure 1b. The pH-dependent modulation of fluorescence intensity was further confirmed with confocal microscopy images in the inset of Figure 1b. To quantify the change in the intensity with pH, we calculated the value of (I(pH) – IpH7.4)/IpH7.4, as shown in Figure 1c, where I(pH) is the PL intensity measured at 577 nm at a given pH and IpH7.4 is the intensity at pH 7.4. As the pH was decreased from 7.4 to 6.6, the 13



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value of (I(pH) – IpH7.4)/IpH7.4 increased from 0 to 1.14. That is, the PL intensity changed dramatically with small variations of pH, demonstrating the high sensitivity of pH measurements in a narrow pH range close to 7.0. To gain deeper insight into the pH-dependent fluorescence response, the hydrodynamic diameter (Hd) of the F-MoS2 NSs at various pH conditions was estimated by dynamic light scattering (DLS) (Figure 1d). Measurements of pristine MoS2 NSs showed double peaks of Hd at 58 nm and 333 nm (regime I), which were invariant with pH change (Figure S10).50, 51 By contrast, the Hd of the F-MoS2 was strongly dependent on pH. At pH 6.0, the average Hd of the F-MoS2 NSs was measured to be 740 nm (regime II). The larger size of the F-MoS2 relative to the pristine MoS2 NSs is attributed to the grafted polymers that are highly stretched in water. We note that the Hd of the F-MoS2 at pH 6.0 from the DLS measurements might not reflect the actual size of the grafted polymers, but is much overestimated one due to the expansion of ionized polymer in aqueous solution.16,

52

Nevertheless, the trend of increasing Hd of F-MoS2 NS with pH was consistent with those of the polymer conformation and the PL spectra. Interestingly, the Hd and its distribution of FMoS2 NSs were comparable to that of the pristine MoS2 NSs at pH 7.0. This is attributed to the collapse of the grafted polymers, which have a very small thickness, yielding almost no change in dimensions. When the pH was increased further to 7.4, the F-MoS2 NSs showed three peaks. Two peaks were located in the regimes I and II with low intensity, whereas one peak was at ~5.5 μm (regime III) at high intensity. This indicates that precipitates of F-MoS2 NSs were dominantly formed in the solution as a result of the significant reduction of solubility of the grafted polymers; free polymers of P20 are insoluble and precipitate. At pH higher than 7.4, the Hd of F-MoS2 NSs was not precisely measured by DLS due to the formation of the large agglomerates caused by the more reduced solubility of the F-MoS2 NSs. The formation of 14


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aggregates at pH 7.4 rendered the dispersion milky red as a result of Mie scattering (Figure S11). These observations indicate that the pH-dependent fluorescent intensity of the F-MoS2 NSs was dictated by the conformational and solubility changes of the grafted polymers. However, it is worth to mention that the F-MoS2 NSs exhibited much better dispersion stability at pH 7.4 and 7.0 than the pristine P20 polymer due to higher dispersion ability of the MoS2 NS. To better understand the mechanism of the fluorescence response of the F-MoS2 system, the average fluorescence lifetimes (τave) of the RB in the F-MoS2 NSs was measured as a function of pH by TRF spectroscopy using the emission at 577 nm upon excitation at 455 nm (Figure S12). The TRF spectra were fitted by a double exponential decay model to obtain the τave values. The τave of the RB in F-MoS2 NSs increased as pH decreased, attributed to P20 stretching. The τave was determined to be 1.9 ns at pH 7.4, and increased to 2.4 ns at pH 6.0 as the emission decay process was prolonged. The increased lifetime of the RB emission at pH 6.0 was attributed to a decrease in the non-radiative decay rate, suggesting suppressed FRET from the dyes to the MoS2.28, 31 However, the FRET efficiency change caused by the coil-toglobule transition of the polymers is not the sole reason for the pH-dependent fluorescence intensity.53-55 The value of (τpH6.0 – τpH7.4)/τpH7.4 is 0.3, which is much smaller than value of (IpH6.0 – IpH7.4)/IpH7.4 = 1.2. This suggests that there is another factor contributing to the steep change of fluorescent intensity with pH. We speculate that the lower solubility of P20 at higher pH leads to lower dispersion stability of F-MoS2 NSs. The formation of aggregates at high pH makes the dispersion opaque, which likely reduces both the absorption of the excitation source by intended absorbers and increases the scattering of the PL emission of F-MoS2 NSs. This socalled ‘inner filter effect’ strengthens the pH dependence of fluorescent intensity.56-59

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3.3 pH Response and PT Effect from F-MoS2 NSs Encapsulated Microcapsules

Figure 2. (a, b) Schematic illustration and OM image of the capillary microfluidic device for generation of monodisperse water-in-oil-in-water (W/O/W) double-emulsion droplets. The monomer-containing oil shell of the double-emulsion droplets is converted to the solid membrane through photopolymerization. (c) Confocal microscopy image of monodisperse microcapsules containing F-MoS2 NSs in the core. (d, e) Confocal microscopy images of the F-MoS2 NSs-loaded microcapsules at pH 7.4 (d) and pH 6.0 (e). Scale bars in (c) and (d, e) are 200 μm and 100 μm, respectively. (f) pH-dependent fluorescence intensity of the microcapsules, where an excitation source with a wavelength of 543 nm was used and each point was averaged from 10 microcapsules.

In common, biological fluids contain adhesive proteins and lipids, which can adsorb to and aggregate the F-MoS2 NSs, and thereby deactivate the pH response. Furthermore, dilution of the F-MoS2 NSs by the fluids reduces the fluorescence intensity, interrupting the 16


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accurate measurement of pH. Therefore, to reliably monitor the local pH of biological fluids, the F-MoS2 NSs were encapsulated within a semipermeable membrane to exclude adhesive molecules and maintain a sufficiently high concentration of F-MoS2 NSs. As a template to produce microcapsules, we used W/O/W double-emulsion droplets. As the inner phase, the aqueous dispersion of the F-MoS2 NSs with the concentration of 0.53 g L-1 at pH 6.0 was used; this dispersion used for the bulk characterization was used without dilution of F-MoS2 NSs or addition of other materials. As the middle phase, a photocurable prepolymer of a polysiloxane modified with methacrylate groups (SB4722) was used. The double-emulsions were prepared with uniform size and composition using a capillary microfluidic device,60-62 comprised of two tapered cylindrical capillaries encased by a square capillary, as shown in Figure 2a. The tapered capillaries with orifice diameters of 120 μm and 240 μm were rendered hydrophobic and hydrophilic, respectively, by surface treatments. These two cylindrical capillaries were arranged within the square capillary to produce a coaxial device by tip-to-tip alignment with a tip separation of 120 μm. An aqueous dispersion of F-MoS2 NSs was injected into the hydrophobic capillary with 120 μm orifice to form the inner water droplets. The prepolymer was injected through the interstices between the hydrophobic capillary and the square capillary to form oil shell.63, 64 In this case, the prepolymer is highly hydrophobic, which prevents the transfer of F-MoS2 NSs from inner water drop to the oil shell and the dissolution of the prepolymers into the inner water drop. In addition, the shell material prepared by photocuring the prepolymers is highly transparent (Figure S13a). Therefore, it is expected that the PL intensity of the F-MoS2 NSs in capsules is not reduced from that in bulk (Figure S13e). In contrast, double emulsions prepared with other prepolymers caused the spontaneous transfer of the F-MoS2 NSs due to the solubility of the pH-responsive poly(DEA-r-BMA) in the prepolymers (Figure S13b-d). Moreover, the polysiloxanes rubber formed by photo-curing 17



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yields membranes with high flexibility and integrity (Figure S14), enabling the injection of the microcapsules for either in vitro or in vivo applications.65-67 As a continuous phase, 10 w/w % aqueous solution of poly(vinyl alcohol) (PVA) was injected through the interstices between the hydrophilic capillary with 240 μm orifice and the square capillary as a counter-flow to the innermost and middle phases; PVA was used to stabilize the outer O/W interfaces. As the innermost, middle, and continuous phases coaxially flow into the 240 μm orifice, doubleemulsion drops are produced through a single-step emulsification in a dripping mode, as shown in Figure 2b. The flow rates of the inner, middle, and continuous phases were set to 500, 135, and 3000 μL h-1, respectively. The monodisperse double-emulsion drops were collected in a distilled water, which was then irradiated with UV light, yielding stable microcapsules. These microcapsules had an average diameter of 235 μm, with a coefficient of variation of 2.2%, indicating high uniformity. The microcapsule membranes had a thickness of approximately 10 μm (Figure S15). The membrane is composed of cross-linked polysiloxane matrix, which retained FMoS2 NSs in the core without a leakage (Figure 2c) and excluded large proteins and lipids. At the same time, the membranes allowed the diffusion of small ions and water molecules. To roughly estimate the required time for pH measurement, microcapsules containing the pHsensitive dye of bromothymol blue were prepared with the same microfluidic method.60-62 The microcapsules were suspended in aqueous solution at pH 6.0, then transferred into a pH 7.0 solution. The microcapsules, which were a yellow color at pH 6.0, gradually turned green upon immersion in the pH 7.0 solution (Figure S16). The color no longer changed after 4 h, which can be considered as the time required for pH measurement of the surrounding solution. To study pH response of the MoS2 NSs in the microcapsules, the loaded microcapsules were incubated in two different solutions, at pH 7.4 and 6.0 for 24 h. The microcapsules at pH 18


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7.4 showed weak fluorescence and those at pH 6.0 produced strong fluorescence, as shown in Figures 2d and e. The fluorescence intensity measured from confocal microscope images showed an abrupt change in the range of pH 7.4 - 6.0, as shown in Figure 2f. This trend is in excellent agreement with that of the F-MoS2 NSs in aqueous solution (Figure 1).

Figure 3. (a) Time-dependent temperature change of suspensions of microcapsules containing F-MoS2 NSs during irradiation with an 808 nm laser at an intensity of 1.0 W cm−2, where three different number concentrations of microcapsules, 2900, 4400, and 7300 units ml-1, were used. For a comparison, distilled water and aqueous solution of P20 were also tested. (b) IR thermal photographs of the microcapsules at 7300 units ml-1 taken at different times during the 808 nm laser irradiation.

The MoS2 NSs are known to have excellent PT properties34-36 and when encapsulated in the microcapsules to prevent dilution, these materials were anticipated to serve as efficient PT heaters. To investigate the PT performance of the microcapsules, we suspended the microcapsules in distilled water at three different number concentrations of 2900, 4400, and 7300 units ml-1 and irradiated them with a laser with a wavelength of 808 nm at an intensity of 1.0 W cm-2. During the irradiation, the temperature increased and eventually reached steady state, as shown in Figure 3a. The steady state temperature increased with the number concentration of microcapsules. With microcapsules at the concentration of 7300 units ml-1, 19



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the temperature increased from 24°C to over 50°C within 5 min, as shown in Figure 3b. This maximum temperature exceeds the critical temperature to ablate the cancer cells.68 The large increase in temperature is attributed to the PT effect of MoS2 NSs; distilled water and an aqueous solution of P20 showed only a marginal increase in temperature (ΔT < 2°C) under the same irradiation conditions. These experiments demonstrate the potential benefits of the MoS2 NS-loaded capsules for localized PT heating.

Figure 4. (a) Schematic illustration showing the in-situ pH monitoring and PT therapy by the microcapsules in a medium containing cancer cells. (b) Series of overlaid bright-field and fluorescence confocal microscopy images (top) and confocal microscopy images (bottom) showing the progressive enhancement of fluorescence intensity in the microcapsules during the growth of A549 tumor cells. (c) The viability of A549 cells in the medium with and without the microcapsules as a function of irradiation time. The cells were incubated in the presence of the microcapsules for 3 days before the irradiation, which showed high cell viability (> 97%). (d, e) OM images of A549 cells before (d) and after (e) irradiation for 20 min. Scale bars in (b) and (d, e) are 200 μm and 50 μm, respectively. 20


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The microcapsules can be potentially used for simultaneous in-situ local pH sensing and PT heating in biological tissues containing a growing population of cancer cells. To demonstrate their use in this context, the F-MoS2 NSs-loaded microcapsules were suspended in an RPMI medium, where adenocarcinomic human alveolar basal epithelial cells (A549) were cultured and monitored over incubation time, as shown in Figure 4a, b. During the first 24 h of incubation (Day 1), the fluorescence intensity of the microcapsules remained low because the external pH was larger than 7.0. On the second day of incubation (Day 2), the fluorescence signal of the microcapsules increased as the pH of the medium changed to 6.6. On the third day (Day 3), the fluorescent intensity increased greatly as the pH of the medium was further decreased to less than or equal to 6.3 by secretion of a lactic acid upon tumor cell growth (Figure 4b and Figure S17). The A549 cells can be ablated by in-situ PT heating under NIR irradiation. In the following experiments, A549 cells were cultured in the presence of the NS-loaded microcapsules at a number concentration of 7300 units ml-1 for 3 days prior to the PT treatment, which showed high viability (> 97%), indicating the high biocompatibility of the microcapsules; in addition, the cell viability was evaluated for various concentrations of the microcapsules, which was higher than 98% for all the concentrations in the range of 0 – 7300 units ml-1 (Figure S18). The microcapsules and A549 cells were then irradiated with an 808 nm laser. The cell viability rapidly decreased to 59%, and the temperature of the cell medium increased to 50 °C after 5 min of irradiation, as shown in Figure 4c. It is known that tumor cell death is usually enacted within several minutes, as cell necrosis is known to occur above 48°C.67 The cell viability dropped to 42% after 10 min of irradiation and to 21% after 20 min. In contrast, irradiation in the absence of microcapsules had no effect on the viability, highlighting the importance of the microcapsules for the selective PT heating. Cell viability 21



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was also visualized after PT heating by treating the cells with 0.4% trypan blue dye (Figure 4d, e). Live cells remained intact and spread on the plate without being stained, whereas dead cells were stained blue and had spherical or stick-like shapes as they contracted and detached from the plate. These results suggest that the microcapsules are promising for efficient PT therapy of cancers. The microcapsules containing the F-MoS2 NSs are potentially useful for in-vivo cancer detection and PT treatment. The feasible route is as follows. The microcapsules suspended in PBS buffer will be subcutaneously injected into suspicious tissues. Relatively large microcapsules may need to be placed in a combination with biopsical or surgical procedures. Once the microcapsules are implanted, the semipermeable membrane of the microcapsules will protect the F-MoS2 NSs from the complex biological fluids and maintain a sufficiently high concentration of F-MoS2 NSs. For the in-vivo uses, the dye with excitation and emission in NIR region is preferred rather than the visible one because of relatively high penetration depth of NIR light along the skin and tissue69, 70, since MoS2 NSs have a broad absorption for visible and NIR range, they would serve as an efficient FRET quencher even for NIR dyes. 29, 34 With the F-MoS2 NSs working at NIR range, the fluorescence intensity can be monitored outside the body. If there were tumors in the implantation sites of the microcapsules, the fluorescence intensity will increase as pH is decreased; otherwise, the intensity will remain unchanged. The sites with increased intensity are then irradiated with a NIR light to photothermally ablate cancer cells. Afterward, the fluorescence intensity from the sites is continuously monitored. The intensity will decrease if the cancer cells were successfully ablated, whereas it will remain unchanged if the cancer cells were not successfully removed.

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4. CONCLUSIONS In summary, we developed a new class of capsule-type pH sensors using MoS2 NSs functionalized with pH-responsive polymers having fluorescent end groups. The pHresponsive polymers were judiciously designed to produce a dramatic change of FRET efficiency in a narrow pH range near 7.0 through optimization of the copolymer composition. The polymer coatings for the NSs provided high dispersion stability and reversible pHresponsive behavior, while the MoS2 NSs served as efficient FRET quenchers. The F-MoS2 NSs with pH-sensitive FRET were microfluidically encapsulated with a hydrophobic, semipermeable membrane using W/O/W double-emulsion droplets as a template. The microcapsules retained the functionalized MoS2 NSs without any leakage or dilution, while enabling in-situ pH meausrements by allowing the diffusion of small ions through the membrane. At the same time, the membranes excluded adhesive molecules in the surrounding solution, which prevented irreversible aggregation and deactivation of the pH-responsive polymer coatings. Further, the PT properties of MoS2 NSs were harnessed to generate remote heating for cancer therapy upon NIR irradiation. With the dual functionalities of pH sensing and PT heating, the microcapsules were successfully used for in-situ pH monitoring in a culture medium of cancer cells and subsequent PT treatment to kill the cells in vitro. The microcapsule sensors are injectable and implantable in target volumes, which further improves the prospects of these materials in biomedical contexts. Moreover, tuning the FRET donors and acceptors to yield fluorescence output in NIR range with a large optical penetration depth in skin and tissue represents an important future direction. In addition, microcapsule arrays are anticipated to allow the analysis of the spatial distribution of pH as each microcapsule reports the pH of the surrounding microenvironment. 23



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ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Characteristics, synthetic scheme and FTIR spectra of poly(DEA-r-BMA)-RB; Phase behaviors of P0-P30 as function of pH; TEM and elemental mapping images of pristine MoS2 and F-MoS2; UV-Vis-NIR and FT-IR spectra of poly(DEA-r-BMA), poly(DEAr-BMA)-RB, F- MoS2; TGA thermograms of MoS2, P20, and P20-coated F-MoS2; Photographs of F-MoS2 NSs and pristine MoS2 NSs in various media; DLS data for pristine MoS2 NSs in pH buffer solutions; Fluorescence decay of F-MoS2 at pH 7.4 and 6.0; Characterization data of microcapsules; Change of pH of cancer medium each cell growth period; Cytotoxicity of F-MoS2 incorporated microcapsules (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] and [email protected] Author Contribution †

These authors contributed equally.

ACKNOWLEDGMENT This work was supported by Samsung Research Funding Center of Samsung Electronics under Project

Number

SRFC-MA1301-07

and

Midcareer

Researcher

Program 24


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(2017R1A2A2A05001156) through the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP). We thank Dr. Rachel Letteri for helpful discussions.

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Microcapsules Containing pH-Responsive, Fluorescent Polymer

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