Article pubs.acs.org/Biomac
Spatiotemporal Monitoring Endocytic and Cytosolic pH Gradients with Endosomal Escaping pH-Responsive Micellar Nanocarriers Jinming Hu, Guhuan Liu, Cheng Wang, Tao Liu, Guoying Zhang, and Shiyong Liu* CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *
ABSTRACT: Endosomal escape is of crucial importance to increase the therapeutic efficacy for nanoparticle-based drug and gene delivery. It has been long presumed that pH-responsive polymeric nanocarriers are potent in aiding endosomal escape due to the “proton sponge” effect; however, the intracellular pH (pHi) gradients subjected by pHresponsive nanocarriers during endocytic and endosomal escaping processes remain to be quantified and elucidated. We herein report the fabrication of ultrasensitive ratiometric fluorescent pHi imaging probes with robust endosomal escaping capability derived from dual dye-labeled pH-responsive block copolymers, which can directly monitor endosomal escape in living cells and quantitatively measure pHi variations during the entire endocytic and endosomolytic processes. Micellar nanoparticle-based pHi sensors could be efficiently internalized into cells via endocytosis where micelle-to-unimer transition occurs, followed by endosomal escape into the cytosol. This process is accompanied by deactivation of blue coumarin emission within acidic organelles and restored blue/red dual emissions within the neutral cytosolic milieu, allowing for ratiometric fluorescent imaging of entire pHi gradients subjected by micellar nanoparticles following the endocytic transport pathway.
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protonation of nanocarriers, and the so-called “proton sponge” effect will help elevate the endosomal escaping efficiency.24 Although enhanced endosomal escape aided by the “proton sponge” effect has been partially supported by some in vitro and in vivo experimental results,25−27 the direct monitoring of pH-actuated endosomal escape of polymeric nanocarriers, and quantifying intracellular pH (pHi) changes subjected by nanocarriers during endocytosis are still lacking. For instance, endosomal escape mediated by “proton sponge” effect is expected to lead to elevated pH within endolysosomes. However, recent studies confirmed that the well-known “proton sponge” polymer, namely poly(ethylene imine) (PEI), cannot induce lysosomal pH changes.28 Therefore, direct imaging and probing of pH varations occurred within endocytic and endosomolytic processes would provide deeper insight into elucidate the endosomal escape mechanism. In this context, although a vast number of fluorescent pHi sensors evolved from small molecule dyes,29−32 fluorescent polymers,33−42 biomacromolecules (e.g., fluorescent proteins),43,44 and nanoparticles45−49 have been explored, they either tend to be retained within specific organelles or evenly distributed through the whole cell. There is still no clear correlation between endosomal escape and endocytic/endo-
INTRODUCTION Nanoparticle-based delivery systems for chemotherapeutic drugs, proteins, vaccine antigens, and genes have emerged as a potent and promising platform for the treatment of cancers and other diseases,1−6 which are capable of targeting and controlled release of therapeutic/diagnostic agents. To date, diverse types of nanocarriers including liposomes,7,8 polymeric micelles and vesicles,3,9−11 and organic/inorganic hybrid nanoparticles12−15 have been developed. Among them, nanostructured aggregates self-assembled from amphiphilic or double hydrophilic block copolymers (DHBCs) have been most frequently explored as delivery vehicles.16−21 However, the therapeutic efficacy of these nanocarriers is proved to be limited due to the presence of several biological barriers.22 Note that in many cases the ideal delivery site is the cytoplasm or the cytosol, but conventional polymeric nanocarriers are typically internalized via endocytosis and follow intracellular endolysosomal trafficking pathway. Thus, they tend to be retained in endosomes and eventually trapped within lysosomes due to insufficient endosomal escaping capability. To boost therapeutic efficacy, it is thus of paramount importance to impart delivery nanocarriers with robust endosomal escape capability.23 To this end, much effort has been devoted to the design of pH-responsive polymeric nanocarriers by taking advantage of the acidic milieu occurred within endosomes and lysosomes. pH gradients within these specific cellular organelles will lead to © 2014 American Chemical Society
Received: September 1, 2014 Revised: October 14, 2014 Published: October 15, 2014 4293
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TMR) were purchased from Invitrogen Company. 7-Hydroxycoumarin-3-carboxylic acid N-succinimidyl ester (NHS-HCCME) was purchased from Aldrich and used as received. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO and used as received. Tetrahydrofuran (THF) was dried by refluxing over sodium shavings and distilled just prior to use. Triethylamine (TEA) was dried by refluxing over CaH2 and distilled prior to use. Nitrate salts (Ag+, Al3+, Ca2+, Cu2+, Mg2+, Pb2+, and Zn2+) were used for all sensing experiments. 1,4-Dioxane and other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ cm. 2-Propylsulfanylthiocarbonyl sulfanyl-2-methyl propionic acid (PTPA) 57,58 and 2-(tertbutoxycarbonylamino)ethyl methacrylamide (BEMA)59 were synthesized according to literature procedures. Sample Synthesis. Synthetic schemes employed for the preparation of HCCME-labeled diblock copolymers (BP1−BP10) and their corresponding precursors are shown in Scheme 2 and Supporting Information Scheme S1. Synthesis of P(DMA-co-APMA) MacroRAFT Agent (Scheme S1). P(DMA-co-APMA) macroRAFT agent was synthesized via RAFT copolymerization of DMA and APMA·HCl monomers. In a typical run, DMA (1.98 g, 20 mmol), PTPA (48 mg, 0.2 mmol), AIBN (3.3 mg, 0.02 mmol), APMA·HCl (36 mg, 0.2 mmol), water (2 mL), and 1,4-dioxane (4 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 70 °C in an oil bath and stirring for 2 h, the reaction tube was terminated by quenching into liquid nitrogen and opened. Water was removed on a rotary evaporator under reduced pressure, and the crude product was diluted with 5 mL 1,4-dioxane. The mixture was then precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated three times to afford yellowish P(DMA-co-APMA) macroRAFT agent (1.2 g, yield = 58.1%; Mn = 7.0 kDa, Mw/Mn = 1.15). The degree of polymerization, DP, was determined to be 72 by 1H NMR analysis in CHCl3 of the crude product before purification. The as-prepared copolymer was thus denoted as P(DMA-co-APMA)72. Synthesis of PS-Based MacroRAFT Agent (Scheme S1). St (2.0 g, 19.2 mmol), PTPA (28.6 mg, 0.12 mmol), AIBN (2.0 mg, 12 μmol), and 1,4-dioxane (2 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 80 °C in an oil bath and stirring for 12 h, the reaction tube was quenched into liquid nitrogen, opened, diluted with CH2Cl2, and then precipitated into an excess of methanol for three times. After drying in a vacuum oven overnight at room temperature, PS macroRAFT agent was obtained as a yellowish powder (0.13 g, yield = 6.4%; Mn,GPC = 2.1 kDa, Mw/Mn = 1.10). The DP of PS macroRAFT agent was determined to be 21 by 1H NMR analysis in CHCl3. The resultant PS macroRAFT agent was thus denoted as PS21. Synthesis of P(DMA-co-APMA)-Based Diblock Copolymers via RAFT Polymerizations Using P(DMA-co-APMA)-Based macroRAFT Agent (Scheme S1). Typically, P(DMA-co-APMA) macromolecular RAFT agent (222 mg, 0.03 mmol), DPA (640 mg, 3.0 mmol), AIBN (0.5 mg, 3 μmol), and 1,4-dioxane (3 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 70 °C in an oil bath and stirring for 4 h, the reaction tube was quenched into liquid nitrogen, opened, and diluted with 3 mL 1,4-dioxane. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 48 h, and then lyophilized as a white solid (468 mg, yield = 54.3%; Mn,GPC = 23.8 kDa, Mw/Mn = 1.18). The conversion of DPA was determined to be 74% based on 1H NMR analysis in CDCl3 of the crude product before purification. The obtained diblock copolymer was therefore denoted as P(DMA-co-APMA)72-b-PDPA74. According to similar procedures, other diblock copolymers were also synthesized.
somolytic pH variations subjected by pH-responsive polymeric nanocarriers. It is thus highly desirable to develop novel pHi sensors capable of spatiotemporally visualizing pHi gradients and variations during endocytosis and subsequent endosomal escaping processes. Inspired by the design of polymeric nanocarriers containing endosomal escaping motifs for enhanced cytosolic delivery,50−56 we envisage that the integration of ratiometric fluorescent pH-sensitive dyes with endosomal escaping pH-responsive block copolymer (BCP) micelles should allow for the sequential fluorescent mapping of intracellular transport pathways and concomitantly endocytic/ cytosolic pH gradients subjected by BCP nanocarriers (Scheme 1). Scheme 1. Construction of a Fluorescent Intracellular Ph Sensor Based on Dual Dye-Labeled P(DMA-co-HCCME)-bP(DEA-co-BMA-co-TMR) Block Copolymer (BP6) Micellesa
a
After cellular internalization, micelle-to-unimer transition occurs within acidic organelles, accompanied with the deactivation of HCCME blue emission; upon endosomal escape and entering into neutral cytosolic milieu, micellar reassembly occurs with restored blue emission. The switchable dual blue/red emission feature allows for ultrasensitive and ratiometric fluorescent monitoring intracellular trafficking pathways.
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MATERIALS AND METHODS
Materials. N,N-Dimethylacrylamide (DMA, Acros), N,N-diethylaminoethyl methacrylate (DEA, Aldrich), butyl methacrylate (BMA, Aldrich, 98%), and methyl methacrylate (MMA, Sinopharm Chemical Reagent Co.) were vacuum-distilled over CaH2 and stored at −20 °C prior to use. Styrene (St, 99.5%, Beijing Chemical Factory) was successively washed with aqueous NaOH (5.0 wt %) and water, and then distilled over CaH2 at reduced pressure. 2-(Diisopropylamino)ethyl methacrylate (DPA) was purchased from Polyscience Company and distilled under reduced pressure prior to use. N-(3-Aminopropyl)methacrylamide hydrochloride (APMA·HCl) and chloroquine diphosphate salt were purchased from Aldrich and used as received. 2,2′Azobis(2-methylpropionitrile) (AIBN) was recrystallized from 95% ethanol. LysoTracker Green and NHS-tetramethylrhodamine (NHS4294
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Scheme 2. Synthetic Routes Employed for the Preparation of Dye-Labeled pH-Responsive Diblock Copolymers (BP1−BP6)
Synthesis of HCCME-Labeled Diblock Copolymers via Amidation of APMA Residuals (Scheme S1). Typically, P(DMA-co-APMA)-b-PDPA (100 mg, 4.3 μmol), NHS-HCCME (2.7 mg, 8.9 μmol), and NEt3 (0.9 mg, 8.9 μmol) were dissolved in 5 mL of anhydrous THF and stirred for 5 h at room temperature. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 48 h, and then lyophilized as a white solid (BP2, 46 mg, yield = 44.8%; Mn,GPC = 23 kDa, Mw/Mn = 1.17, Table S1). HCCME content in P(DMA-co-HCCME) block was determined to be ∼0.56 mol % by fluorescence spectroscopy in ethanol by using NHSHCCME as the calibration standard. According to the similar procedures, other HCCME-labeled diblock copolymers (BP1 and BP3−BP9) were also synthesized, and the structural parameters are summarized in Table S1. Synthesis of P(DMA-co-HCCME)-b-P(DEA-co-BMA-co-TMR) (Scheme 2, BP10). P(DMA-co- HCCME)72-b-P(DEA0.705-coBMA0.285-co-BEMA0.01)67 (50 mg, 2.6 μmol) and 0.5 mL of TFA were dissolved in 0.5 mL of CH2Cl2 and stirred overnight at room temperature. After removing all the solvent, NHS-TMR (2.7 mg, 5.0 μmol) and NEt3 (0.9 mg, 8.9 μmol) were dissolved in 10 mL of anhydrous THF and added. The mixture was stirred for 5 h at room temperature. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 24 h and then lyophilized as a red solid (BP10, 26 mg, yield = 49.3%; Mn,GPC = 19.9 kDa, Mw/Mn = 1.17, Table S1). TMR content in P(DEA-co-BMA-co-TMR) block was determined to be ∼1.0 mol % by fluorescence spectroscopy in ethanol by using NHS-TMR as the calibration standard. Fabrication of Diblock Copolymer Micelles. Self-assembled micelles from diblock copolymers were prepared via the cosolvent approach. In a typical run, BP3 (5 mg) was dissolved in 1 mL of DMF. Then the solution was added into 8.0 mL of deionized water under vigorous stirring over ∼1 h. Then the polymer solution was dialyzed against deionized water for 24 h (MWCO 3.5 kDa), and fresh deionized water was replaced every 6 h. Finally, the colloidal dispersions were diluted to the desired concentrations for further experiments. In Vitro Cytotoxicity Assay. HepG2 cells were employed for in vitro cytotoxicity evaluation via the MTT assay. HepG2 cells were first cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a CO2−air (5:95) incubator for 2 days. For the cytotoxicity assay, HeLa cells were seeded in a 96-well plate at an initial density of ca. 5000 cells/well in 100 μL of complete DMEM medium. After incubating for 24 h, DMEM was replaced with fresh medium, and the cells were treated with polymer solution at varying final concentrations. The treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 48 h. The MTT reagent (5.0 g/L, in PBS) was added 20 μL to each well. The cells were further incubated for 4 h at 37 °C. The medium in each well was then removed and replaced by 150 μL of DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each
experiment was conducted in quadruple and the data are shown as the mean value plus a standard deviation (±SD). Hemolysis Assay. The endosomal escape capability of the copolymers was assessed by hemolysis assay conducted according to literature procedures.60,61 Briefly, polymers (P1 and BP1−BP9) with varying concentrations were incubated for 1 h at 37 °C in the presence of human red blood cells (RBC) in 100 mM sodium phosphate buffers (supplemented with 150 mM NaCl) of varying pH (7.4, 7.0, 6.5, 6.0, and 5.5) mimicking the acidifying pH gradient of endosomes at different phases. PBS and Triton X-100 (1% v/v) were employed as negative and positive hemolysis controls, respectively. After centrifugation at 1000 rpm for 10 min, the absorbance at 405 nm of the supernatant was recorded by using a microplate reader (Thermo Fisher). The hemolytic activity was defined as H = (A − A0)/(ATX − A0) where A denotes the absorbance reading of the sample well, A0 denotes the negative control, and ATX denotes the positive control. Cell Culture and in Vitro Fluorescence Imaging. HepG2 cells (∼105) in DMEM complete medium were plated onto 35 mm uncoated, glass-bottomed culture dishes and incubated overnight. Cells were then exposed to HCCME-labeled polymers ([HCCME] = 1.0 × 10−6 M) at 37 °C for up to 2 h in DMEM complete medium. Cells were rinsed with PBS (3 × 1 mL) and DMEM complete medium before incubating for up to an additional 2, 6, 14, and 22 h at 37 °C. Fluorescence images of labeled cells and controls were acquired using an inverted Leica SP2 confocal microscope at 37 °C. The samples were excited at 405 nm for HCCME, 488 nm for LysoTracker Green, and 543 nm for TMR, and the fluorescence was collected between 430 and 470 nm for the blue channel, 510−550 nm for the green channel, and 555−595 nm for the red channel, respectively. All confocal laser scanning microscopy (CLSM) images were taken under the same conditions for parallel comparison. For chloroquine coincubation experiments, chloroquine (100 μM, pH 7.4) in DMEM was added into the culture dishes and the cells were incubated for an additional 1 h before imaging. To adjust intracellular pH, NH4Cl aqueous solution (30 mM NH4Cl and 30 mM NaCl) and PBS were used, respectively. The intracellular calibration curve used for determining intracellular pH was obtained by calculating the fluorescence intensity ratio of BP10 at blue and red channels using nigericin and varying pH buffers according to literature procedures.62,63 Characterization. All 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H) operated in the Fourier transform mode. CDCl3 was used as the solvent. Molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with Waters 1515 pump and Waters 2414 differential refractive index detector (set at 30 °C), employing a series of two linear Styragel columns (HR2 and HR4) at an oven temperature of 45 °C. The eluent was DMF at a flow rate of 1.0 mL/ min. A series of low polydispersity polystyrene standards were employed for calibration. The hydrodynamic diameters and zeta potentials of the self-assembled micelles in PBS (10 mM, pH 7.4) were 4295
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determined by Malvern Zetasizer Nano ZS. All data were averaged over three consecutive measurements and the polymer concentration was fixed at 1.0 g/L. Transmission electron microscopy (TEM) observations were conducted on a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV. Fluorescence spectra were recorded on F-4600 (Hitachi) spectrofluorometer. The fluorescence images of HepG2 cells were acquired using inverted Leica SP2 confocal microscope at 37 °C.
sensing capability of HCCME-labeled BCPs, the pH-dependent emission of HCCME-conjugated precursor (P1) was evaluated at first. Under neutral or alkaline media, aqueous P1 solution possesses intense blue emission when excited at 405 nm, whereas the emission intensity dramatically decreases upon lowering pH (Figures 1a and S2). Quantitative analysis revealed
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RESULTS AND DISCUSSION Characterization of Fluorescent Dye-Labeled pHResponsive Micellar Nanocarriers. We start from the synthesis of a series of BCPs (BP1−BP6) consisting of hydrophilic PDMA block covalently conjugated with 7hydroxycoumarin fluorophores (HCCME), which are capable of fluorescent pH sensing,64,65 and pH-responsive P(DEA-coBMA) block endowing endosomal escaping features,51,52 where DMA, DEA, and BMA are N,N-dimethylacrylamide, N,Ndiethylaminoethyl methacrylate, and n-butyl methacrylate, respectively (Schemes 2 and S1, Table 1). It is wellTable 1. Molecular Parameters of Dye-Labeled Polymer Precursors and Diblock Copolymers entry
samples
P1 BP1
P(DMA-co-HCCME)72 P(DMA-co-HCCME)72-bP(DEA0.933-co-BMA0.067)74 P(DMA-co-HCCME)72-bP(DEA0.818-co-BMA0.182)76 P(DMA-co-HCCME)72-bP(DEA0.699-co-BMA0.301)69 P(DMA-co-HCCME)72-bP(DEA0.608-co-BMA0.392)74 P(DMA-co-HCCME)72-bP(DEA0.502-co-BMA0.498)66 P(DMA-co-HCCME)72-bP(DEA0.705-co-BMA0.285-coTMR0.01)67 P(DMA-co-HCCME)72-b-PMMA80 P(DMA-co-HCCME)72-b-PDPA74 P(DMA-co-HCCME)65-b-PS21 P(DMA-co-HCCME)72-b-PDEA76
BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 a b
Mn,GPC (kDa)a
Mw/Mna
Mn,NMR (kDa)b
7.1 20.5
1.14 1.18
7.4 21.0
20.7
1.14
21.0
19.7
1.15
19.4
19.8
1.15
20.0
18.5
1.16
18.3
19.9
1.16
20.0
15.8 23.0 9.7 21.5
1.19 1.17 1.14 1.17
15.5 23.3 8.9 21.6
Figure 1. Normalized (a) fluorescence emission spectra (λex = 405 nm) and (b) relative intensity changes (λem = 447 nm) recorded for P1 aqueous solution in the range of pH 2−10. HCCME moieties are nonfluorescent at acidic pH but highly fluorescent at neutral pH. Normalized (c) emission spectra (λex = 405 nm for HCCME and λex = 543 nm for TMR) and (d) emission intensity ratio changes (Iblue/Ired) recorded for BP6 in the range of pH 2−10. Iblue and Ired refer to intensity integrations between 420−470 nm and 555−595 nm, respectively.
a cumulative ∼1153-fold emission change at 447 nm in the pH range of 2−10. Notably, most of the fluorescence changes occurs within a relatively narrow pH range (i.e., pH 4−8) (Figure 1b). pH-dependent changes in P1 fluorescence originate from the blue shift of excitation peak maximum (405 nm at pH 10 and 350 nm at pH 2) with an isoexcitation point at ∼368 nm (Figures S3). When excited at 405 and 368 nm, respectively, I(λ405)/I(λ368) exhibited ∼1134-fold change in the pH range of 2−10. It should be noted that this impressive performance is far more superior to that of most previously reported pH probes,33−37,68−70 indicating that P1 could serve as a self-calibrated pH sensor with ratiometric fluorescent detection mode (Figure S4). In addition, the P1 emission was not affected by common bioactive molecules, metal ions, and enzymes (Figure S5). Thus, HCCME-labeled polymer should be eligible for full range pHi sensing. Upon incubating HepG2 cells with small molecule HCCME, it was found that HCCME dye cannot be efficiently taken up by cells; moreover, the addition of lysosomotropic agent (chloroquine) did not lead to apparent fluorescence emission enhancement, suggesting poor membrane permeability of HCCME dye (Figure S6). In contrast, for HCCME-labeled P1 copolymer, weak blue emission could be observed after 24 h incubation (Figure S6), whereas 2 h incubation resulted in negligible emission. Intriguingly, intense blue emission was clearly discernible even for 2 h incubation in the presence of chloroquine. This suggested that the internalized P1 was mainly located within acidic organelles (e.g., endosomes and lysosomes) and chloroquine addition can not only inhibit endolysosomal acidification but also promote endosome escape of P1 into the neutral cytosol, which synergistically contribute to the increased blue emission.50,71,72 We then reasoned that
Determined by GPC using DMF as the eluent (1.0 mL/min). Calculated from 1H NMR results.
documented that PDEA (pKa ∼ 7.0) is hydrophilic at pH < 7 and turns hydrophobic at pH higher than the pKa.66 Accordingly, BP1−BP6 possess pH-actuated micellization behavior in aqueous media (Table S1). For example, BP6 self-assembles into micelles with pH-responsive P(DEA-coBMA) cores at pH 7.4, exhibiting an intensity-average hydrodynamic diameter, ⟨Dh⟩, of ∼24.2 nm, as revealed by dynamic light scattering (DLS) (Figure S1a). The formation of micelles was further verified by TEM observations (Figure S1b). Potentiometric titrations revealed that the pKa of BP1− BP5 BCPs gradually decreases with increasing BMA contents due to a shift of hydrophobic/hydrophilic balance (Table S1). Note that the pKa range (6.73−5.57) of BP1−BP5 is consistent with endolysosomal pH in living cells, which should facilitate the endosomolytic process and help bypass the endolysosomal pathway,67 allowing for the entire fluorescent pHi sensing of endocytic and cytosolic pH gradients. Extracellular and Intracellular Fluorescence Studies of pH-Responsive Micellar Nanocarriers. To assess the pH 4296
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Figure 2. Confocal laser scanning microscopy (CLSM) images of live HepG2 cells after incubating at 37 °C for varying durations with (a) BP1, (b) BP2, (c) BP3, (d) BP4, (e) BP5, (f) BP7, (g) BP8, and (h) BP9. HCCME concentrations were fixed at 1.0 × 10−6 M in all cases.
Figure 3. (A) Incubation duration-dependent CLSM images of live HepG2 cells when culturing with BP6 copolymer at 37 °C. Incubation durationdependent (B) Normalized blue and red channel emission intensities quantified from confocal microscopy measurements. (C) Evolution of pH gradient distributions subjected by BP6 nanocarriers during endocytic trafficking.
assays revealed that BP10 was the most cytotoxic especially at high concentrations; therefore, it was excluded for intracellular imaging studies (Figure S8). BP1−BP5 and BP7−BP9 were then coincubated with HepG2 cells and their pHi sensing performance was checked (Figure 2). Typically, upon extending incubation time, blue emission can be observed within cells for BP1−BP5 with varying BMA contents (Figure 2a−e). In sharp contrast, blue emissions were nondiscernible for BP7−BP9 (serving as control BCPs) even after 24 h coincubation (Figure
although the emission intensity of P1 decreases with pH decrease (Figure 1b), it might serve as an excellent fluorescence turn-on indicator for monitoring endosomal escaping capability of nanocarriers. To verify this hypothesis, HCCME-labeled BCPs (BP1−BP5 and BP7−BP10) with varying chemical structures/compositions and different endosomal escaping tendency were examined (Table 1). Just like P1, all BCP micellar nanoparticles underwent pH-switchable emission (Figure S7). Cell viability 4297
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Figure 4. (a) Incubation duration-dependent CLSM images of live HepG2 cells when culturing at 37 °C with BP6 and LysoTracker Green. (b) Incubation duration-dependent normalized red channel emission intensities quantified from CLSM measurements. (C) Colocalization ratio analysis between red channel fluorescence from TMR moieties and green channel fluorescence of stained endolysosomes (LysoTracker Green). The green channel was excited at 488 nm and collected between 510 and 550 nm, and the red channel was excited at 543 nm and collected between 555 and 595 nm.
fluorescently monitoring the whole endocytic process and pHi gradients. To solve this issue, we then opted to conjugate pHinert dye, tetramethylrhodamine (i.e., TMR), onto the pHresponsive block. TMR can serve as an internal standard and enable ratiometric fluorescent pH imaging by using cofocal microscopy.35,74 At pH 10, the micellar solution of dual dye (HCCME and TMR)-labeled BP6 exhibited both red emission at 575 nm and blue emission at 447 nm (Figure 1c). Upon increasing solution pH, the emission peak at ∼447 nm substantially increased, yet the emission at 575 nm exhibited a moderate enhancement. We infer that the enhanced emission at 575 nm should be ascribed to fluorescence resonance energy transfer (FRET) between coumarin and TMR moieties when considering the pH-inert feature of TMR dye.75 This result also corroborated with the formation of micellar nanocarriers upon pH increase, thereby decreasing the spatial distance between different types of fluorophores. Specifically, in the pH range of 2−10, emission intensity ratios (I447/I575) exhibited a cumulative ∼125-fold increase, with most of the changes having occurred within the pH range of 4−8 (Figure 1d). Moreover, pH-regulated changes in emission intensity ratios were fully reversible when the solution pH was cycled between 4 and 9 (Figure S12). Endocytic pH Evolution Imaging and the Endosomal Escape Mechanism of pH-Responsive Micellar Nanocarriers. Next, the pHi imaging performance of BP6 copolymer was conducted (Figure 3). The TMR red channel emission could be detected during the entire incubation period due to its always-on nature. In contrast, the blue channel HCCME emission was almost nondiscernible for the first 4 h incubation; whereas after 8 h, blue emission can be discerned and steadily
2f−h). These results demonstrated that the introduction of pHresponsive amphiphilic P(DEA-co-BMA) block poses a great advantage for endosomal escape, which is in accord with previous literature reports.51,52,73 Quantitative analysis revealed that BP3 (∼30 mol % BMA in the responsive block) exhibited the maximum emission enhancement (∼100-fold increase) after 24 h incubation (Figure S9). The endosomolytic capability was further corroborated by hemolysis experiments, indicating that BP3 induced the most significant hemolysis effect (Figure S10). Since the BMA content of BP6 copolymer is quite close to that of BP3, we can safely assume that BP6 should exhibit comparable endosomal escaping capability. Note that the coincubation with BP7−BP9 for 24 h resulted in negligible blue emission (Figure 2f−h); this might be ascribed to either poor cellular uptake and/or trapping within acidic organelles. To probe this issue, endolysomolytic agent was introduced and the coincubation with chloroquine only for 2 h can significantly enhance HCCME blue emissions for all BCPs (BP1−BP5 and BP7−BP9; Figure S11). This suggested that all BCP nanocarriers can be efficiently internalized by HepG2 cells and the negligible blue emission for BP7−BP9 without chloroquine should be due to accumulation within acidic organelles. For BP1−BP5, pH-induced micelle-to-unimer transition within acidic organelles and the amphiphilic nature of protonated P(DEA-co-BMA) block synergistically contribute to endosomal escape.56 Although HCCME-labeled BCPs possess built-in ratiometric fluorescent pH sensing feature when excited at 405 and 368 nm (Figure S4), the UV laser line is less available in conventional CLSM setup. In addition, HCCME moieties exhibit quenched emission within acidic milieu, and it is thus not suitable for 4298
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Figure 5. (a−e) CLSM images recorded for live HepG2 cells incubated at 37 °C for 24 h with BP6 at varying conditions. (f) Starvation and (g) addition of NH4Cl can induce pHi decrease and increase, respectively. Further addition of DMEM can restore pHi to the original level and pHi variations can be cycled for several times. The blue channel was excited at 405 nm and collected between 420 and 470 nm, and the red channel was excited at 543 nm and collected between 555 and 595 nm. The data in (f) and (g) represent a single experiment performed in triplicate with the error bars denoting the standard deviation.
increased upon extended incubation (Figure 3A and B). The emerging blue emission implied that BP6 can spontaneously escape from endosomes and enter into the cytosol. This is reasonable considering that the BMA content in BP6 is quite comparable to that in BP3, which possesses the most prominent endosomolytic tendency. On the basis of pH calibration curve constructed via an established method (Figure S13),62,63 quantitative analysis clearly suggested a pH shift of local milieu subjected by BP6 nanocarriers during endocytic trafficking. Within the first 8 h, the nanocarriers were probing a local pH of ∼5; upon 16 and 24 h incubation, the local milieu gradually shifted to around neutral pH (Figure 3C). This transition is also in accordance with the sequential endocytic trafficking pathway, that is, internalization into acidic organelles and endosomal escape into the neutral cytosol (Scheme 1). To further verify the intracellular transport pathway and endosomolytic feature of BP6-based pHi sensor, the acidic organelles were costained with LysoTracker Green. BP6 micelles were initially located within acidic endosomes for the first 4 h, as evidenced by the overlay between green-emissive LysoTracker Green and red-emissive TMR (Figure 4). This is also in agreement with the nondiscernible blue emission of HCCME moieties (Figure 3a). For an incubation duration over 8 h, red fluorescence originating from TMR moieties was observed, indicating that micellar nanocarriers have increasingly escaped from acidic organelles. Moreover, extensive endosomal escape was verified by the colocalization ratio between green and red channels, decreasing from ∼92% for 4 h incubation to
Spatiotemporal Monitoring Endocytic and Cytosolic pH Gradients with Endosomal Escaping pH-Responsive Micellar Nanocarriers Jinming Hu, Guhuan Liu, Cheng Wang, Tao Liu, Guoying Zhang, and Shiyong Liu* CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *
ABSTRACT: Endosomal escape is of crucial importance to increase the therapeutic efficacy for nanoparticle-based drug and gene delivery. It has been long presumed that pH-responsive polymeric nanocarriers are potent in aiding endosomal escape due to the “proton sponge” effect; however, the intracellular pH (pHi) gradients subjected by pHresponsive nanocarriers during endocytic and endosomal escaping processes remain to be quantified and elucidated. We herein report the fabrication of ultrasensitive ratiometric fluorescent pHi imaging probes with robust endosomal escaping capability derived from dual dye-labeled pH-responsive block copolymers, which can directly monitor endosomal escape in living cells and quantitatively measure pHi variations during the entire endocytic and endosomolytic processes. Micellar nanoparticle-based pHi sensors could be efficiently internalized into cells via endocytosis where micelle-to-unimer transition occurs, followed by endosomal escape into the cytosol. This process is accompanied by deactivation of blue coumarin emission within acidic organelles and restored blue/red dual emissions within the neutral cytosolic milieu, allowing for ratiometric fluorescent imaging of entire pHi gradients subjected by micellar nanoparticles following the endocytic transport pathway.
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protonation of nanocarriers, and the so-called “proton sponge” effect will help elevate the endosomal escaping efficiency.24 Although enhanced endosomal escape aided by the “proton sponge” effect has been partially supported by some in vitro and in vivo experimental results,25−27 the direct monitoring of pH-actuated endosomal escape of polymeric nanocarriers, and quantifying intracellular pH (pHi) changes subjected by nanocarriers during endocytosis are still lacking. For instance, endosomal escape mediated by “proton sponge” effect is expected to lead to elevated pH within endolysosomes. However, recent studies confirmed that the well-known “proton sponge” polymer, namely poly(ethylene imine) (PEI), cannot induce lysosomal pH changes.28 Therefore, direct imaging and probing of pH varations occurred within endocytic and endosomolytic processes would provide deeper insight into elucidate the endosomal escape mechanism. In this context, although a vast number of fluorescent pHi sensors evolved from small molecule dyes,29−32 fluorescent polymers,33−42 biomacromolecules (e.g., fluorescent proteins),43,44 and nanoparticles45−49 have been explored, they either tend to be retained within specific organelles or evenly distributed through the whole cell. There is still no clear correlation between endosomal escape and endocytic/endo-
INTRODUCTION Nanoparticle-based delivery systems for chemotherapeutic drugs, proteins, vaccine antigens, and genes have emerged as a potent and promising platform for the treatment of cancers and other diseases,1−6 which are capable of targeting and controlled release of therapeutic/diagnostic agents. To date, diverse types of nanocarriers including liposomes,7,8 polymeric micelles and vesicles,3,9−11 and organic/inorganic hybrid nanoparticles12−15 have been developed. Among them, nanostructured aggregates self-assembled from amphiphilic or double hydrophilic block copolymers (DHBCs) have been most frequently explored as delivery vehicles.16−21 However, the therapeutic efficacy of these nanocarriers is proved to be limited due to the presence of several biological barriers.22 Note that in many cases the ideal delivery site is the cytoplasm or the cytosol, but conventional polymeric nanocarriers are typically internalized via endocytosis and follow intracellular endolysosomal trafficking pathway. Thus, they tend to be retained in endosomes and eventually trapped within lysosomes due to insufficient endosomal escaping capability. To boost therapeutic efficacy, it is thus of paramount importance to impart delivery nanocarriers with robust endosomal escape capability.23 To this end, much effort has been devoted to the design of pH-responsive polymeric nanocarriers by taking advantage of the acidic milieu occurred within endosomes and lysosomes. pH gradients within these specific cellular organelles will lead to © 2014 American Chemical Society
Received: September 1, 2014 Revised: October 14, 2014 Published: October 15, 2014 4293
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TMR) were purchased from Invitrogen Company. 7-Hydroxycoumarin-3-carboxylic acid N-succinimidyl ester (NHS-HCCME) was purchased from Aldrich and used as received. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO and used as received. Tetrahydrofuran (THF) was dried by refluxing over sodium shavings and distilled just prior to use. Triethylamine (TEA) was dried by refluxing over CaH2 and distilled prior to use. Nitrate salts (Ag+, Al3+, Ca2+, Cu2+, Mg2+, Pb2+, and Zn2+) were used for all sensing experiments. 1,4-Dioxane and other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ cm. 2-Propylsulfanylthiocarbonyl sulfanyl-2-methyl propionic acid (PTPA) 57,58 and 2-(tertbutoxycarbonylamino)ethyl methacrylamide (BEMA)59 were synthesized according to literature procedures. Sample Synthesis. Synthetic schemes employed for the preparation of HCCME-labeled diblock copolymers (BP1−BP10) and their corresponding precursors are shown in Scheme 2 and Supporting Information Scheme S1. Synthesis of P(DMA-co-APMA) MacroRAFT Agent (Scheme S1). P(DMA-co-APMA) macroRAFT agent was synthesized via RAFT copolymerization of DMA and APMA·HCl monomers. In a typical run, DMA (1.98 g, 20 mmol), PTPA (48 mg, 0.2 mmol), AIBN (3.3 mg, 0.02 mmol), APMA·HCl (36 mg, 0.2 mmol), water (2 mL), and 1,4-dioxane (4 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 70 °C in an oil bath and stirring for 2 h, the reaction tube was terminated by quenching into liquid nitrogen and opened. Water was removed on a rotary evaporator under reduced pressure, and the crude product was diluted with 5 mL 1,4-dioxane. The mixture was then precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated three times to afford yellowish P(DMA-co-APMA) macroRAFT agent (1.2 g, yield = 58.1%; Mn = 7.0 kDa, Mw/Mn = 1.15). The degree of polymerization, DP, was determined to be 72 by 1H NMR analysis in CHCl3 of the crude product before purification. The as-prepared copolymer was thus denoted as P(DMA-co-APMA)72. Synthesis of PS-Based MacroRAFT Agent (Scheme S1). St (2.0 g, 19.2 mmol), PTPA (28.6 mg, 0.12 mmol), AIBN (2.0 mg, 12 μmol), and 1,4-dioxane (2 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 80 °C in an oil bath and stirring for 12 h, the reaction tube was quenched into liquid nitrogen, opened, diluted with CH2Cl2, and then precipitated into an excess of methanol for three times. After drying in a vacuum oven overnight at room temperature, PS macroRAFT agent was obtained as a yellowish powder (0.13 g, yield = 6.4%; Mn,GPC = 2.1 kDa, Mw/Mn = 1.10). The DP of PS macroRAFT agent was determined to be 21 by 1H NMR analysis in CHCl3. The resultant PS macroRAFT agent was thus denoted as PS21. Synthesis of P(DMA-co-APMA)-Based Diblock Copolymers via RAFT Polymerizations Using P(DMA-co-APMA)-Based macroRAFT Agent (Scheme S1). Typically, P(DMA-co-APMA) macromolecular RAFT agent (222 mg, 0.03 mmol), DPA (640 mg, 3.0 mmol), AIBN (0.5 mg, 3 μmol), and 1,4-dioxane (3 mL) were charged into a glass ampule equipped with a magnetic stirring bar. The glass ampule was carefully degassed via three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 70 °C in an oil bath and stirring for 4 h, the reaction tube was quenched into liquid nitrogen, opened, and diluted with 3 mL 1,4-dioxane. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 48 h, and then lyophilized as a white solid (468 mg, yield = 54.3%; Mn,GPC = 23.8 kDa, Mw/Mn = 1.18). The conversion of DPA was determined to be 74% based on 1H NMR analysis in CDCl3 of the crude product before purification. The obtained diblock copolymer was therefore denoted as P(DMA-co-APMA)72-b-PDPA74. According to similar procedures, other diblock copolymers were also synthesized.
somolytic pH variations subjected by pH-responsive polymeric nanocarriers. It is thus highly desirable to develop novel pHi sensors capable of spatiotemporally visualizing pHi gradients and variations during endocytosis and subsequent endosomal escaping processes. Inspired by the design of polymeric nanocarriers containing endosomal escaping motifs for enhanced cytosolic delivery,50−56 we envisage that the integration of ratiometric fluorescent pH-sensitive dyes with endosomal escaping pH-responsive block copolymer (BCP) micelles should allow for the sequential fluorescent mapping of intracellular transport pathways and concomitantly endocytic/ cytosolic pH gradients subjected by BCP nanocarriers (Scheme 1). Scheme 1. Construction of a Fluorescent Intracellular Ph Sensor Based on Dual Dye-Labeled P(DMA-co-HCCME)-bP(DEA-co-BMA-co-TMR) Block Copolymer (BP6) Micellesa
a
After cellular internalization, micelle-to-unimer transition occurs within acidic organelles, accompanied with the deactivation of HCCME blue emission; upon endosomal escape and entering into neutral cytosolic milieu, micellar reassembly occurs with restored blue emission. The switchable dual blue/red emission feature allows for ultrasensitive and ratiometric fluorescent monitoring intracellular trafficking pathways.
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MATERIALS AND METHODS
Materials. N,N-Dimethylacrylamide (DMA, Acros), N,N-diethylaminoethyl methacrylate (DEA, Aldrich), butyl methacrylate (BMA, Aldrich, 98%), and methyl methacrylate (MMA, Sinopharm Chemical Reagent Co.) were vacuum-distilled over CaH2 and stored at −20 °C prior to use. Styrene (St, 99.5%, Beijing Chemical Factory) was successively washed with aqueous NaOH (5.0 wt %) and water, and then distilled over CaH2 at reduced pressure. 2-(Diisopropylamino)ethyl methacrylate (DPA) was purchased from Polyscience Company and distilled under reduced pressure prior to use. N-(3-Aminopropyl)methacrylamide hydrochloride (APMA·HCl) and chloroquine diphosphate salt were purchased from Aldrich and used as received. 2,2′Azobis(2-methylpropionitrile) (AIBN) was recrystallized from 95% ethanol. LysoTracker Green and NHS-tetramethylrhodamine (NHS4294
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Scheme 2. Synthetic Routes Employed for the Preparation of Dye-Labeled pH-Responsive Diblock Copolymers (BP1−BP6)
Synthesis of HCCME-Labeled Diblock Copolymers via Amidation of APMA Residuals (Scheme S1). Typically, P(DMA-co-APMA)-b-PDPA (100 mg, 4.3 μmol), NHS-HCCME (2.7 mg, 8.9 μmol), and NEt3 (0.9 mg, 8.9 μmol) were dissolved in 5 mL of anhydrous THF and stirred for 5 h at room temperature. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 48 h, and then lyophilized as a white solid (BP2, 46 mg, yield = 44.8%; Mn,GPC = 23 kDa, Mw/Mn = 1.17, Table S1). HCCME content in P(DMA-co-HCCME) block was determined to be ∼0.56 mol % by fluorescence spectroscopy in ethanol by using NHSHCCME as the calibration standard. According to the similar procedures, other HCCME-labeled diblock copolymers (BP1 and BP3−BP9) were also synthesized, and the structural parameters are summarized in Table S1. Synthesis of P(DMA-co-HCCME)-b-P(DEA-co-BMA-co-TMR) (Scheme 2, BP10). P(DMA-co- HCCME)72-b-P(DEA0.705-coBMA0.285-co-BEMA0.01)67 (50 mg, 2.6 μmol) and 0.5 mL of TFA were dissolved in 0.5 mL of CH2Cl2 and stirred overnight at room temperature. After removing all the solvent, NHS-TMR (2.7 mg, 5.0 μmol) and NEt3 (0.9 mg, 8.9 μmol) were dissolved in 10 mL of anhydrous THF and added. The mixture was stirred for 5 h at room temperature. The polymer solution was purified by dialysis (MWCO 3.5 kDa) against deionized water for 24 h and then lyophilized as a red solid (BP10, 26 mg, yield = 49.3%; Mn,GPC = 19.9 kDa, Mw/Mn = 1.17, Table S1). TMR content in P(DEA-co-BMA-co-TMR) block was determined to be ∼1.0 mol % by fluorescence spectroscopy in ethanol by using NHS-TMR as the calibration standard. Fabrication of Diblock Copolymer Micelles. Self-assembled micelles from diblock copolymers were prepared via the cosolvent approach. In a typical run, BP3 (5 mg) was dissolved in 1 mL of DMF. Then the solution was added into 8.0 mL of deionized water under vigorous stirring over ∼1 h. Then the polymer solution was dialyzed against deionized water for 24 h (MWCO 3.5 kDa), and fresh deionized water was replaced every 6 h. Finally, the colloidal dispersions were diluted to the desired concentrations for further experiments. In Vitro Cytotoxicity Assay. HepG2 cells were employed for in vitro cytotoxicity evaluation via the MTT assay. HepG2 cells were first cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a CO2−air (5:95) incubator for 2 days. For the cytotoxicity assay, HeLa cells were seeded in a 96-well plate at an initial density of ca. 5000 cells/well in 100 μL of complete DMEM medium. After incubating for 24 h, DMEM was replaced with fresh medium, and the cells were treated with polymer solution at varying final concentrations. The treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 48 h. The MTT reagent (5.0 g/L, in PBS) was added 20 μL to each well. The cells were further incubated for 4 h at 37 °C. The medium in each well was then removed and replaced by 150 μL of DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each
experiment was conducted in quadruple and the data are shown as the mean value plus a standard deviation (±SD). Hemolysis Assay. The endosomal escape capability of the copolymers was assessed by hemolysis assay conducted according to literature procedures.60,61 Briefly, polymers (P1 and BP1−BP9) with varying concentrations were incubated for 1 h at 37 °C in the presence of human red blood cells (RBC) in 100 mM sodium phosphate buffers (supplemented with 150 mM NaCl) of varying pH (7.4, 7.0, 6.5, 6.0, and 5.5) mimicking the acidifying pH gradient of endosomes at different phases. PBS and Triton X-100 (1% v/v) were employed as negative and positive hemolysis controls, respectively. After centrifugation at 1000 rpm for 10 min, the absorbance at 405 nm of the supernatant was recorded by using a microplate reader (Thermo Fisher). The hemolytic activity was defined as H = (A − A0)/(ATX − A0) where A denotes the absorbance reading of the sample well, A0 denotes the negative control, and ATX denotes the positive control. Cell Culture and in Vitro Fluorescence Imaging. HepG2 cells (∼105) in DMEM complete medium were plated onto 35 mm uncoated, glass-bottomed culture dishes and incubated overnight. Cells were then exposed to HCCME-labeled polymers ([HCCME] = 1.0 × 10−6 M) at 37 °C for up to 2 h in DMEM complete medium. Cells were rinsed with PBS (3 × 1 mL) and DMEM complete medium before incubating for up to an additional 2, 6, 14, and 22 h at 37 °C. Fluorescence images of labeled cells and controls were acquired using an inverted Leica SP2 confocal microscope at 37 °C. The samples were excited at 405 nm for HCCME, 488 nm for LysoTracker Green, and 543 nm for TMR, and the fluorescence was collected between 430 and 470 nm for the blue channel, 510−550 nm for the green channel, and 555−595 nm for the red channel, respectively. All confocal laser scanning microscopy (CLSM) images were taken under the same conditions for parallel comparison. For chloroquine coincubation experiments, chloroquine (100 μM, pH 7.4) in DMEM was added into the culture dishes and the cells were incubated for an additional 1 h before imaging. To adjust intracellular pH, NH4Cl aqueous solution (30 mM NH4Cl and 30 mM NaCl) and PBS were used, respectively. The intracellular calibration curve used for determining intracellular pH was obtained by calculating the fluorescence intensity ratio of BP10 at blue and red channels using nigericin and varying pH buffers according to literature procedures.62,63 Characterization. All 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H) operated in the Fourier transform mode. CDCl3 was used as the solvent. Molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with Waters 1515 pump and Waters 2414 differential refractive index detector (set at 30 °C), employing a series of two linear Styragel columns (HR2 and HR4) at an oven temperature of 45 °C. The eluent was DMF at a flow rate of 1.0 mL/ min. A series of low polydispersity polystyrene standards were employed for calibration. The hydrodynamic diameters and zeta potentials of the self-assembled micelles in PBS (10 mM, pH 7.4) were 4295
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determined by Malvern Zetasizer Nano ZS. All data were averaged over three consecutive measurements and the polymer concentration was fixed at 1.0 g/L. Transmission electron microscopy (TEM) observations were conducted on a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV. Fluorescence spectra were recorded on F-4600 (Hitachi) spectrofluorometer. The fluorescence images of HepG2 cells were acquired using inverted Leica SP2 confocal microscope at 37 °C.
sensing capability of HCCME-labeled BCPs, the pH-dependent emission of HCCME-conjugated precursor (P1) was evaluated at first. Under neutral or alkaline media, aqueous P1 solution possesses intense blue emission when excited at 405 nm, whereas the emission intensity dramatically decreases upon lowering pH (Figures 1a and S2). Quantitative analysis revealed
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RESULTS AND DISCUSSION Characterization of Fluorescent Dye-Labeled pHResponsive Micellar Nanocarriers. We start from the synthesis of a series of BCPs (BP1−BP6) consisting of hydrophilic PDMA block covalently conjugated with 7hydroxycoumarin fluorophores (HCCME), which are capable of fluorescent pH sensing,64,65 and pH-responsive P(DEA-coBMA) block endowing endosomal escaping features,51,52 where DMA, DEA, and BMA are N,N-dimethylacrylamide, N,Ndiethylaminoethyl methacrylate, and n-butyl methacrylate, respectively (Schemes 2 and S1, Table 1). It is wellTable 1. Molecular Parameters of Dye-Labeled Polymer Precursors and Diblock Copolymers entry
samples
P1 BP1
P(DMA-co-HCCME)72 P(DMA-co-HCCME)72-bP(DEA0.933-co-BMA0.067)74 P(DMA-co-HCCME)72-bP(DEA0.818-co-BMA0.182)76 P(DMA-co-HCCME)72-bP(DEA0.699-co-BMA0.301)69 P(DMA-co-HCCME)72-bP(DEA0.608-co-BMA0.392)74 P(DMA-co-HCCME)72-bP(DEA0.502-co-BMA0.498)66 P(DMA-co-HCCME)72-bP(DEA0.705-co-BMA0.285-coTMR0.01)67 P(DMA-co-HCCME)72-b-PMMA80 P(DMA-co-HCCME)72-b-PDPA74 P(DMA-co-HCCME)65-b-PS21 P(DMA-co-HCCME)72-b-PDEA76
BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 a b
Mn,GPC (kDa)a
Mw/Mna
Mn,NMR (kDa)b
7.1 20.5
1.14 1.18
7.4 21.0
20.7
1.14
21.0
19.7
1.15
19.4
19.8
1.15
20.0
18.5
1.16
18.3
19.9
1.16
20.0
15.8 23.0 9.7 21.5
1.19 1.17 1.14 1.17
15.5 23.3 8.9 21.6
Figure 1. Normalized (a) fluorescence emission spectra (λex = 405 nm) and (b) relative intensity changes (λem = 447 nm) recorded for P1 aqueous solution in the range of pH 2−10. HCCME moieties are nonfluorescent at acidic pH but highly fluorescent at neutral pH. Normalized (c) emission spectra (λex = 405 nm for HCCME and λex = 543 nm for TMR) and (d) emission intensity ratio changes (Iblue/Ired) recorded for BP6 in the range of pH 2−10. Iblue and Ired refer to intensity integrations between 420−470 nm and 555−595 nm, respectively.
a cumulative ∼1153-fold emission change at 447 nm in the pH range of 2−10. Notably, most of the fluorescence changes occurs within a relatively narrow pH range (i.e., pH 4−8) (Figure 1b). pH-dependent changes in P1 fluorescence originate from the blue shift of excitation peak maximum (405 nm at pH 10 and 350 nm at pH 2) with an isoexcitation point at ∼368 nm (Figures S3). When excited at 405 and 368 nm, respectively, I(λ405)/I(λ368) exhibited ∼1134-fold change in the pH range of 2−10. It should be noted that this impressive performance is far more superior to that of most previously reported pH probes,33−37,68−70 indicating that P1 could serve as a self-calibrated pH sensor with ratiometric fluorescent detection mode (Figure S4). In addition, the P1 emission was not affected by common bioactive molecules, metal ions, and enzymes (Figure S5). Thus, HCCME-labeled polymer should be eligible for full range pHi sensing. Upon incubating HepG2 cells with small molecule HCCME, it was found that HCCME dye cannot be efficiently taken up by cells; moreover, the addition of lysosomotropic agent (chloroquine) did not lead to apparent fluorescence emission enhancement, suggesting poor membrane permeability of HCCME dye (Figure S6). In contrast, for HCCME-labeled P1 copolymer, weak blue emission could be observed after 24 h incubation (Figure S6), whereas 2 h incubation resulted in negligible emission. Intriguingly, intense blue emission was clearly discernible even for 2 h incubation in the presence of chloroquine. This suggested that the internalized P1 was mainly located within acidic organelles (e.g., endosomes and lysosomes) and chloroquine addition can not only inhibit endolysosomal acidification but also promote endosome escape of P1 into the neutral cytosol, which synergistically contribute to the increased blue emission.50,71,72 We then reasoned that
Determined by GPC using DMF as the eluent (1.0 mL/min). Calculated from 1H NMR results.
documented that PDEA (pKa ∼ 7.0) is hydrophilic at pH < 7 and turns hydrophobic at pH higher than the pKa.66 Accordingly, BP1−BP6 possess pH-actuated micellization behavior in aqueous media (Table S1). For example, BP6 self-assembles into micelles with pH-responsive P(DEA-coBMA) cores at pH 7.4, exhibiting an intensity-average hydrodynamic diameter, ⟨Dh⟩, of ∼24.2 nm, as revealed by dynamic light scattering (DLS) (Figure S1a). The formation of micelles was further verified by TEM observations (Figure S1b). Potentiometric titrations revealed that the pKa of BP1− BP5 BCPs gradually decreases with increasing BMA contents due to a shift of hydrophobic/hydrophilic balance (Table S1). Note that the pKa range (6.73−5.57) of BP1−BP5 is consistent with endolysosomal pH in living cells, which should facilitate the endosomolytic process and help bypass the endolysosomal pathway,67 allowing for the entire fluorescent pHi sensing of endocytic and cytosolic pH gradients. Extracellular and Intracellular Fluorescence Studies of pH-Responsive Micellar Nanocarriers. To assess the pH 4296
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Figure 2. Confocal laser scanning microscopy (CLSM) images of live HepG2 cells after incubating at 37 °C for varying durations with (a) BP1, (b) BP2, (c) BP3, (d) BP4, (e) BP5, (f) BP7, (g) BP8, and (h) BP9. HCCME concentrations were fixed at 1.0 × 10−6 M in all cases.
Figure 3. (A) Incubation duration-dependent CLSM images of live HepG2 cells when culturing with BP6 copolymer at 37 °C. Incubation durationdependent (B) Normalized blue and red channel emission intensities quantified from confocal microscopy measurements. (C) Evolution of pH gradient distributions subjected by BP6 nanocarriers during endocytic trafficking.
assays revealed that BP10 was the most cytotoxic especially at high concentrations; therefore, it was excluded for intracellular imaging studies (Figure S8). BP1−BP5 and BP7−BP9 were then coincubated with HepG2 cells and their pHi sensing performance was checked (Figure 2). Typically, upon extending incubation time, blue emission can be observed within cells for BP1−BP5 with varying BMA contents (Figure 2a−e). In sharp contrast, blue emissions were nondiscernible for BP7−BP9 (serving as control BCPs) even after 24 h coincubation (Figure
although the emission intensity of P1 decreases with pH decrease (Figure 1b), it might serve as an excellent fluorescence turn-on indicator for monitoring endosomal escaping capability of nanocarriers. To verify this hypothesis, HCCME-labeled BCPs (BP1−BP5 and BP7−BP10) with varying chemical structures/compositions and different endosomal escaping tendency were examined (Table 1). Just like P1, all BCP micellar nanoparticles underwent pH-switchable emission (Figure S7). Cell viability 4297
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Figure 4. (a) Incubation duration-dependent CLSM images of live HepG2 cells when culturing at 37 °C with BP6 and LysoTracker Green. (b) Incubation duration-dependent normalized red channel emission intensities quantified from CLSM measurements. (C) Colocalization ratio analysis between red channel fluorescence from TMR moieties and green channel fluorescence of stained endolysosomes (LysoTracker Green). The green channel was excited at 488 nm and collected between 510 and 550 nm, and the red channel was excited at 543 nm and collected between 555 and 595 nm.
fluorescently monitoring the whole endocytic process and pHi gradients. To solve this issue, we then opted to conjugate pHinert dye, tetramethylrhodamine (i.e., TMR), onto the pHresponsive block. TMR can serve as an internal standard and enable ratiometric fluorescent pH imaging by using cofocal microscopy.35,74 At pH 10, the micellar solution of dual dye (HCCME and TMR)-labeled BP6 exhibited both red emission at 575 nm and blue emission at 447 nm (Figure 1c). Upon increasing solution pH, the emission peak at ∼447 nm substantially increased, yet the emission at 575 nm exhibited a moderate enhancement. We infer that the enhanced emission at 575 nm should be ascribed to fluorescence resonance energy transfer (FRET) between coumarin and TMR moieties when considering the pH-inert feature of TMR dye.75 This result also corroborated with the formation of micellar nanocarriers upon pH increase, thereby decreasing the spatial distance between different types of fluorophores. Specifically, in the pH range of 2−10, emission intensity ratios (I447/I575) exhibited a cumulative ∼125-fold increase, with most of the changes having occurred within the pH range of 4−8 (Figure 1d). Moreover, pH-regulated changes in emission intensity ratios were fully reversible when the solution pH was cycled between 4 and 9 (Figure S12). Endocytic pH Evolution Imaging and the Endosomal Escape Mechanism of pH-Responsive Micellar Nanocarriers. Next, the pHi imaging performance of BP6 copolymer was conducted (Figure 3). The TMR red channel emission could be detected during the entire incubation period due to its always-on nature. In contrast, the blue channel HCCME emission was almost nondiscernible for the first 4 h incubation; whereas after 8 h, blue emission can be discerned and steadily
2f−h). These results demonstrated that the introduction of pHresponsive amphiphilic P(DEA-co-BMA) block poses a great advantage for endosomal escape, which is in accord with previous literature reports.51,52,73 Quantitative analysis revealed that BP3 (∼30 mol % BMA in the responsive block) exhibited the maximum emission enhancement (∼100-fold increase) after 24 h incubation (Figure S9). The endosomolytic capability was further corroborated by hemolysis experiments, indicating that BP3 induced the most significant hemolysis effect (Figure S10). Since the BMA content of BP6 copolymer is quite close to that of BP3, we can safely assume that BP6 should exhibit comparable endosomal escaping capability. Note that the coincubation with BP7−BP9 for 24 h resulted in negligible blue emission (Figure 2f−h); this might be ascribed to either poor cellular uptake and/or trapping within acidic organelles. To probe this issue, endolysomolytic agent was introduced and the coincubation with chloroquine only for 2 h can significantly enhance HCCME blue emissions for all BCPs (BP1−BP5 and BP7−BP9; Figure S11). This suggested that all BCP nanocarriers can be efficiently internalized by HepG2 cells and the negligible blue emission for BP7−BP9 without chloroquine should be due to accumulation within acidic organelles. For BP1−BP5, pH-induced micelle-to-unimer transition within acidic organelles and the amphiphilic nature of protonated P(DEA-co-BMA) block synergistically contribute to endosomal escape.56 Although HCCME-labeled BCPs possess built-in ratiometric fluorescent pH sensing feature when excited at 405 and 368 nm (Figure S4), the UV laser line is less available in conventional CLSM setup. In addition, HCCME moieties exhibit quenched emission within acidic milieu, and it is thus not suitable for 4298
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Biomacromolecules
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Figure 5. (a−e) CLSM images recorded for live HepG2 cells incubated at 37 °C for 24 h with BP6 at varying conditions. (f) Starvation and (g) addition of NH4Cl can induce pHi decrease and increase, respectively. Further addition of DMEM can restore pHi to the original level and pHi variations can be cycled for several times. The blue channel was excited at 405 nm and collected between 420 and 470 nm, and the red channel was excited at 543 nm and collected between 555 and 595 nm. The data in (f) and (g) represent a single experiment performed in triplicate with the error bars denoting the standard deviation.
increased upon extended incubation (Figure 3A and B). The emerging blue emission implied that BP6 can spontaneously escape from endosomes and enter into the cytosol. This is reasonable considering that the BMA content in BP6 is quite comparable to that in BP3, which possesses the most prominent endosomolytic tendency. On the basis of pH calibration curve constructed via an established method (Figure S13),62,63 quantitative analysis clearly suggested a pH shift of local milieu subjected by BP6 nanocarriers during endocytic trafficking. Within the first 8 h, the nanocarriers were probing a local pH of ∼5; upon 16 and 24 h incubation, the local milieu gradually shifted to around neutral pH (Figure 3C). This transition is also in accordance with the sequential endocytic trafficking pathway, that is, internalization into acidic organelles and endosomal escape into the neutral cytosol (Scheme 1). To further verify the intracellular transport pathway and endosomolytic feature of BP6-based pHi sensor, the acidic organelles were costained with LysoTracker Green. BP6 micelles were initially located within acidic endosomes for the first 4 h, as evidenced by the overlay between green-emissive LysoTracker Green and red-emissive TMR (Figure 4). This is also in agreement with the nondiscernible blue emission of HCCME moieties (Figure 3a). For an incubation duration over 8 h, red fluorescence originating from TMR moieties was observed, indicating that micellar nanocarriers have increasingly escaped from acidic organelles. Moreover, extensive endosomal escape was verified by the colocalization ratio between green and red channels, decreasing from ∼92% for 4 h incubation to
