Movement of a Quantum Dot Covered with Cytocompatible and pH

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Movement of a quantum dot covered with cytocompatible and pHresponsible phospholipid polymer chains under cellular environment Yihua Liu, Haruka Oda, Yuuki Inoue, and Kazuhiko Ishihara Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01357 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 31, 2016

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Movement of a quantum dot covered with cytocompatible and pHresponsible phospholipid polymer chains under cellular environment Yihua Liu1, Haruka Oda2, Yuuki Inoue1, Kazuhiko Ishihara1,2*

1

Department of Bioengineering and 2Department of Materials Engineering, School of

Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*To whom all correspondence should be addressed: K. Ishihara: e-mail: [email protected]

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ABSTRACT Quantum dots (QDs) were functionalized with well-defined polymer chains having both cytocompatibility and pH-responsiveness to monitor the movement of nanoparticles in a cellular environment with changing local pH. We used a triblock-type water-soluble polymer composed of three segments: (1) pH-responsive poly[2-(N, N-diethylamino)ethyl methacrylate; DEAEMA] segment, (2) poly[ω-(p-nitrophenyloxycarbonyl oligo(ethylene glycol)) methacrylate; MEONP] segment bearing an active ester group to react with an amino compound, and (3) cytocompatible poly[(2-methacryloyloxyethyl phosphorylcholine; MPC) segment. Moreover, hydrophobic and carboxyl groups were attached as terminals of the polymer chain. The triblock-type polymer was attached to the QD surface through a hydrophobic layer, which was covered with the QD by hydrophobic interaction. This produced hybrid QD particles (QD/MPC polymer nanoparticles). The QD/MPC polymer nanoparticles had good water-dispersion ability after the modification. Fluorescence resonance energy transfer (FRET) phenomenon between QD and fluorescence dye (Alexa) was clearly observed at pH 7.4 and 9.0 when a fluorescence dye was reacted with the poly(MEONP) segment of the polymer. However, the efficiency decreased at pH 5.0. This was due to a change in the distance between the QD and the fluorescence dye in response to the protonation degree of the poly(DEAEMA) segment. The permeability of QD/MPC polymer nanoparticles through the cell membrane was enhanced by reacting the cellpenetrating peptide, octaarginine (R8), to the carboxyl group at the end of the polymer. The R8-QD/MPC polymer/Alexa nanoparticles attached onto the HeLa cell membrane surface within 15 min after they were added to the cell culture. This attachment initiated nanoparticle penetration of the cell membrane by endocytosis. The nanoparticles could be followed continuously as they moved in the cell culture. The change in the FRET index was determined during this process. Use of the R8-QD/MPC polymer/Alexa nanoparticle enabled


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us to determine nanoparticle location, based on the surrounding local pH. We concluded that QDs, modified with a cytocompatible and pH-responsible MPC polymer, provide a new imaging and transport tool in cell-based science and engineering.

KEYWORDS: triblock-type phospholipid polymer; surface modification; pH responsibility; fluorescence resonance energy transfer; endosome

INTRODUCTION Semiconductor nanoparticles, quantum dots (QDs) have excellent fluorescence characteristics such as high fluorescence quantum yield, multi-colored fluorescence, and less photoinduced bleaching1-8. Thus, they are highly amenable for application in the biology and medicine fields as fluorescence imaging reagents9-12. However, some drawbacks of QDs, such as cytotoxicity, low dispersion ability, and instability in an aqueous medium2,

13-18

,

limited their application to a biological environment. Hydrophobic compounds such as octadecylamine (ODA) and trioctylphosphine oxide (TOPO)19 are added to form the outermost layer during the QD preparation process. The aforementioned limitations were mainly attributed to the hydrophobic layer of QDs. Therefore, surface modifications to improve the hydrophilic character have been carried out using some detergents and hydrophilic polymers such as poly(ethylene oxide). We previously reported a method for modifying QDs with excellent cytocompatibility. A water-soluble phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units was used to obtain QD/MPC polymer hybrid nanoparticles20-27. The optical properties of QDs were maintained after modification with the MPC polymer. The water-dispersion ability and cytocompatibility were dramatically improved. Development of new applications for QDs in the cellular


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environment may be achieved by introducing additional functions in the MPC polymers, such as pH-responsibility and easy conjugation of specific biomolecules on the surface of QDs27. In this study, we prepared a novel type of QD/MPC polymer hybrid nanoparticles for monitoring both their location and intracellular pH. The triblock-type polymer was synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization to regulate the molecular structures28,29. The strategies for the polymer synthesis were to: (1) obtain benefits from the monomer compositions: poly(2-(N, N-diethylamino)ethyl methacrylate (DEAEMA)) segment, poly(MEONP) segment for binding special molecules, and hydrophilic and cytocompatible poly(MPC) segment; and (2) profit from the terminal hydrophobic alkyl group and terminal carboxyl group derived from the RAFT reagent, 4cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (RAFT19), to immobilize the MPC polymer to the hydrophobic layer on the QDs surfaces30,31 and immobilize the second biomolecule, respectively. Next, a fluorescent dye was immobilized to the MPC polymer via the active ester groups of the poly(MEONP) segment. The pH-responsive poly(DEAEMA) segment could induce a stretching-shrinking conformation in response to the pH of the medium, which provides a nice dynamic platform for the fluorescence resonance energy transfer (FRET) phenomenon between the QD and the fluorescent dye27. The cellular uptake and internalization of QD/MPC polymer nanoparticles were achieved when the cellpenetrating peptide (CPP)32 was immobilized at the terminal carboxyl group. We continuously observed changes in the FRET index during the transport of the QD/MPC polymer nanoparticle into and within the cells.

EXPERIMENTAL SECTION

Materials


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MPC, purchased from NOF Co. Ltd., Tokyo, Japan, was synthesized by a previously reported method33. 2, 2ʹ-Azobisisobutyronitrile (AIBN) was purchased from Kanto Chemical Co. Ltd. (Tokyo, Japan). RAFT19, DEAEMA, and octaarginine (R8) were purchased from Sigma-Aldrich Co. LLC (St. Louis, USA). The DEAEMA was purified by distillation under reduced pressure and fractions at 80°C/10 mmHg were used. The ODA-coated ZnS-shell CdSe-core QDs (excitation wavelength (Ex)/emission wavelength (Em) = 520 nm/535 nm), dispersed in toluene, were purchased from NN-labs, LLC, Fayetteville, NC, USA. Alexa Fluor 568 cadaverine (Alexa) (Ex/Em = 578 nm/602 nm) was obtained from Molecular Probes (Invitrogen, Eugene, WA, USA). The MEONP was synthesized by a previously described method

27,34

. Dulbecco’s phosphate buffered saline (DPBS) was purchased from

Gibco (Invitrogen, Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and lactose dehydrogenase (LDH)-Cytotoxic Test Wako were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Other reagents and solvents were of extra pure grade and used without further purification.

Synthesis of triblock-type polymer The synthetic scheme for the triblock-type MPC polymer, poly(DEAEMA-blockMEONP-block-MPC) (PDbNbM) is described in Figure 1, and Figure S-1 shows a more detailed synthetic scheme. First, the poly(MPC) segment as macroinitiator was synthesized by RAFT polymerization. The MPC (15 mmol) was dissolved in 30 mL of ethanol at a concentration of 0.50 mol/L with the initiator AIBN (0.060 mmol) and RAFT19 (0.30 mmol). The concentration ratios of [MPC]/[RAFT19] and [RAFT19]/[AIBN] were 50 and 5.0, respectively. The mixture was placed in a glass tubing, bubbling with argon (Ar) gas to


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remove oxygen; the glass tubing was sealed. Polymerization was performed at 60°C for 24 h. The unreacted monomers were removed by 2 precipitations from chloroform followed by 1 from diethyl ether. The precipitated polymer was collected by filtration and dried overnight under vacuum to completely evaporate the remaining solvents. Next, the second segment of macroinitiator poly(MEONP-block-MPC) (PNbM) was synthesized. Poly(MPC) macroinitiator (0.16 mmol), MEONP (2.4 mmol), and AIBN (0.030 mmol) were dissolved in 8.0 mL of ethanol at a monomer concentration of 0.30 mol/L. The concentration

ratios

of

[MEONP]/[poly(MPC)

macroinitiator]

and

[poly(MPC)

macroinitiator]/[AIBN] were 15 and 5.0, respectively. The polymerization was performed at 60°C for 20 h. The reacted solution was diluted with ethanol and added dropwise into an excess amount of diethyl ether/chloroform mixture (70/30 by volume) to precipitate the polymer. Triblock-type polymer PDbNbM, composed of poly(DEAEMA) and PNbM segments, was synthesized by RAFT polymerization with PNbM (0.060 mmol), DEAEMA (3.0 mmol), and the initiator AIBN (0.020 mmol) in 10 mL of ethanol. The concentration of DEAEMA was 0.30 mol/L. The concentration ratios of [DEAEMA]/[PNbM] and [PNbM]/[AIBN] were 51 and 3.0, respectively. The polymerization was performed at 60°C for 24 h. Hexane was used to precipitate the formed polymer. The precipitate was filtered and dried overnight under vacuum. The chemical structures of poly(MPC), PNbM, and PDbNbM were analyzed in ethanol-d6, using 1H-NMR spectroscopy (JNM-GX300, JEOL, Tokyo, Japan). The weightaverage molecular weight (Mw) and number-average molecular weight (Mn) of the polymer were determined by gel-permeation chromatography (GPC, JASCO Co. Ltd., Tokyo, Japan). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) containing 10 mmol/L of sodium trifluoroacetate (TFA-Na) was used as the eluent at a flow rate of 0.50 mL/min. The concentration of the


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polymers was 10 mg/mL. Poly(methyl methacrylate (MMA)) standard samples were used to calibrate the molecular weight of the polymer. A very similar procedure was used to synthesize the triblock-type MPC polymer with other poly(DEMEMA) segment lengths to understand the effect of the poly(DEAEMA) segment on pH-responsibility.

Measurement of surface tension of PDbNbM aqueous solutions The PDbNbM aqueous solution (pH ca. 5.5) surface tensions were measured in the range of polymer concentrations between 0.001 mg/mL and 10 mg/mL. The measurement was performed via the Wilhelmy plate method of dynamic contact angle goniometer (DCA100 contact-angle tensiometer, Orientec, Tokyo, Japan) at room temperature (ca. 25°C), using the platinum plate. The measurements were repeated three times for each condition.

Preparation of QD nanoparticles modified with PDbNbM Surface

modification

of

QD

nanoparticles

with

PDbNbM

(QD/PDbNbM

nanoparticles) was carried out by a solvent evaporation method27, 35. The QD purchased stock (100 µL, stored in toluene at 5.0 mg/mL) was drawn into a glass tubing. Toluene was removed under reduced pressure by careful rotary evaporation. The QD was suspended again in 1.0 mL of dichloromethane. The QD solution was added dropwise into 10 mL of PDbNbM aqueous solution (2.5 mg/mL) at room temperature. The mixture was sonicated for 10 min in an ice bath, using an ultrasonic generator (Sonifier 250; Branson, Danbury, CT, USA). The mixture was maintained under reduced pressure for 20 min to evaporate the dichloromethane. The formed nanoparticles that were dispersed in the aqueous solution were collected by ultracentrifugation at 50,000 rpm (Optima L-70k, Beckman Coulter, Palo Alto, CA, USA) for


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2 h at 4°C. A stock suspension was prepared by suspending the QD/PDbNbM nanoparticles in 1.0 mL of water and purified by filtration through a 0.22 µm-pore diameter filter. The elemental compositions of the QD/PDbNbM nanoparticle surface were determined by X-ray photoelectron spectroscopy (XPS, AXIS-HIS, Kratos-Shimadzu, Kyoto, Japan). An Alexa fluorescence dye was introduced to the QD/PDbNbM nanoparticle by the polymer conjugation method. First, the PDbNbM was reacted with the Alexa dye, and then, the QD were modified with the PDbNbM/Alexa. The amino group in the Alexa dye was reacted with the active ester group of the poly(MEONP) segment. The Alexa dye was dissolved in PBS (pH = 8.5) to a concentration of 4.0 µg/mL and the PDbNbM (15 mg/mL) was added to the solution. The mixture was rotated overnight at room temperature. The unreacted Alexa dye was removed by dialysis against water for 2 days. The PDbNbM/Alexa was obtained after freeze-drying for 2 days. Next, the QD surfaces were modified with PDbNbM/Alexa via the solvent evaporation method. The procedure was the same as described for the preparation of QD/PDbNbM nanoparticles.

Fluorescence spectroscopic measurement of QD/PDbNbM/Alexa nanoparticles The QD/PDbNbM/Alexa nanoparticles were suspended in aqueous media with a pH equal to 5.0, 7.4, or 9.0. The fluorescence spectra of the nanoparticle solutions at different pH values were measured by fluorescence spectroscopy (FP, FP8500, Jasco, Tokyo, Japan) at 450 nm. The fluorescence intensity ratio between Alexa and QD was calculated as the FRET index.

Immobilization of cell-penetrating peptide on the QD/PDbNbM/Alexa nanoparticle


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The immobilization reaction began with DMT-MM. The concentration of R8 was 0.080 mmol/L (i.e. 0.10 mg/mL) in the nanoparticle solution, and DMT-MM was 10 mmol/L. A 1.0 mL QD/PDbNbM/Alexa nanoparticle solution was mixed with DMT-MM and R8 overnight. The carboxyl group at the terminal of the polymer chain (originated from RAFT19) reacted with the amino group from the cell-penetrating peptide R8. Unreacted R8 was removed by ultracentrifugation at 50,000 rpm for 2 h at 4°C. The R8QD/PDbNbM/Alexa nanoparticles were suspended again in 1.0 mL PBS.

Cytotoxicity test by lactate dehydrogenase (LDH) activity measurement Cytotoxicity was determined by the amount of LDH that was released from the cells. HeLa cells were seeded on a 96-well cell culture plate (2.0 × 103 cells/well) containing DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. They were incubated under 5% CO2 at 37°C. The cell culture medium was removed after a 20 h incubation. The sample solutions, in 200-µL volumes, were added to the wells and incubated. The samples included R8-QD/PDbNbM/Alexa nanoparticles, QD/PDbNbM/Alexa nanoparticles, and polymers such as PDbNbM/Alexa, PDbNbM, and Alexa. From the value of (weight of QD)/(volume of QD × density of QD)(catalog data from supplier and expected structure of the ODA-coated ZnS-shell CdSe-core QD), we considered that about 2.0 × 1013 nanoparticles were present in 1.0 mL aqueous suspension as a stock suspension. The 100 µL of the suspension was added to the 900 µL cell culture medium to prepare a high concentration nanoparticle suspension. Additionally, another 1/10 dilution was carried out to prepare a low concentration nanoparticle suspension. The negative control was Tween 20 (0.50 vol%) solution, and the positive control was only DMEM. After the sample mixture was incubated with cells for 2 h, 100 µL of the sample solution from the cell culturing well was reacted with 100 µL of the LDH color


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reagent for 25 min. An equal volume (200 µL) of the reaction terminator was used to stop the reaction. The visible light spectrophotometric measurement of LDH was determined at 560 nm. The samples without cells were also tested by measurement of LDH at the same condition. The results showed the value close to 0. Thus, self-absorbance of QD and Alexa did not interfere with the LDH results.

Cellular uptake of R8-QD/PDbNbM/Alexa nanoparticles The cellular uptake of R8-QD/PDbNbM/Alexa nanoparticles was detected with a fluorescence microscope (IX71, Olympus, Tokyo, Japan). HeLa cells were cultured under 5% CO2 for 20 h at 37°C in a 3.5-cm diameter dish containing DMEM (2.0 × 104 cells/cm2). The R8-QD/PDbNbM/Alexa nanoparticle solution (20 µL) was added to the HeLa cells and incubated at 37°C. The progress of cell uptake during the incubation was captured by taking a picture every 15 min after addition of the nanoparticles to the solution. The FRET of R8-QD/PDbNbM/Alexa nanoparticles during the cellular uptake was detected with a confocal laser scanning microscopy (CLSM, FV1000-D IX81, Olympus, Tokyo, Japan). HeLa cells were cultured for 20 h under 5% CO2 at 37°C, in a glass-bottom dish containing DMEM (2.0 × 104 cells/cm2). The nucleus of cells was stained with Hoechst. Ten microliters of the R8-QD/PDbNbM/Alexa nanoparticle solution was added to the HeLa cell culture medium, and incubated at 37°C under 5% CO2. The cell uptake progress was captured by taking pictures at 473 nm every 15 min from the onset of nanoparticle addition until 2.5 h of incubation. The velocity of the R8-QD/PDbNbM/Alexa nanoparticles that moved into the cells was calculated from the CLSM images, using ImageJ (Wayne Rasbund, National Institute of Health, Bethesda, MD, USA http://imagej.nih.gov/ij) with manual tracking plugin (Fabrice Corde at lires, Institut Curie, Orsay, France). The effect of R8QD/PDbNbM/Alexa nanoparticles entrapped in the endosome region was captured during a 1


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h movie from the beginning of the incubation with nanoparticles. The FRET index of R8QD/PDbNbM/Alexa nanoparticles was evaluated in the following procedure. Each fluorescent image was taken with an excitation wavelength of 488 nm and emission wavelength of 488 nm and 566 nm, respectively. The fluorescence intensity at each wavelength in the 100% and 0% FRET index was measured in a water solution at pH 7.4 and pH 5.0 prior to the cellular experiment. The FRET index of R8-QD/PDbNbM/Alexa particles was then evaluated using Metamorph (Molecular Device Japan Co., Tokyo, Japan) by comparison to the values obtained. The regions with a FRET index of less than 0.30 were regarded as “non-FRET” in this study. Finally, the fluorescent pictures were binarized to the “FRET” and “non-FRET” region. Each region was evaluated for its size and numbers, using ImageJ.

RESULTS AND DISCUSSION

Synthesis and characterization of the PDbNbM The results of the PDbNbM synthesis are summarized in Table 1. The chemical structures of the polymers were confirmed by NMR spectroscopy (supporting information Figure. S-2, S-3, S-4). RAFT polymerization not only controlled the molecular weight of the polymers, but it also introduced reactive carboxyl and hydrophobic dodecyl groups at the end of the polymer chain. The poly(MPC) macroinitiator and PNbM were water-soluble and amphiphilic in nature, based on the poly(MPC) segment and immobilization of functional compounds to the poly(MEONP) segment 21. The triblock-type polymer, PDbNbM, was also water-soluble and it comprised 22 DEAEMA units in the poly(DEAEMA) segment, 14 MEONP units in the poly(MEONP) segment, and 50 MPC units in the poly(MPC) segment.


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These results enabled us to conclude that a well-structured triblock-type polymer was obtained. Figure 2 shows the relationship between the surface tension of the PDbNbM aqueous solution and the PDbNbM concentrations. The surface tension of the PDbNbM solution was dependent on the polymer concentration; it decreased with a polymer concentration of approximately 0.040 mg/mL. This result indicated aggregation of PDbNbM in the medium. Stable aggregations were obtained at polymer concentration of approximately 2.5 mg/mL. The surface tension of the poly(MPC) aqueous solution was maintained above 70 mN/m in this range of polymer concentration, owing to its extremely hydrophilic nature27. Therefore, the terminal dodecyl group and the poly(DEAEMA) segment had a hydrophobic nature, which induced aggregation of PDbNbM in an aqueous medium. The stable PDbNbM aggregate was useful for preparing hybrid nanoparticles with QD nanoparticles through a hydrophobic interaction.

Surface elemental composition of QD/PDbNbM nanoparticles Figure 3 shows schematic representation of the QD/PDbNbM hybrid nanoparticle structure. QDs were solubilized into the PDbNbM aggregate during the preparation of QD/PDbNbM nanoparticles using a solvent evaporation method. The terminal hydrophobic group in PDbNbM chains interacted with the hydrophobic ODA layer covering the QDs surface. This hybridization was stable even when the QD/PDbNbM nanoparticles were purified by ultracentrifugation. The water dispersion of the nanoparticles was improved; thus, we obtained a transparent aqueous dispersion of the nanoparticles with an original fluorescence of QD. The modification was confirmed by XPS analysis of the nanoparticles, as shown in Figure 4. XPS signals were observed at 285.0 eV and 405.0 eV in the original QDs. These signals were assigned to the carbon atoms of the alkyl group and the cadmium


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atom, respectively. In contrast, the QD/PDbNbM showed XPS signals at 288.0 eV, 286.5 eV, and 285.0 eV in the carbon atom region. These signals were attributed to the carbon atoms in the carbonyl, ester, and alkyl groups, respectively. We observed signals at 402.3 eV, 399.0 eV, and 133.6 eV, which corresponded to the nitrogen atoms of the amino groups, nitrogen atoms of the ammonium groups, and the phosphorus atom of phosphate group, respectively. These signals were specific for the MPC polymer chains. The XPS signal at 405.0 eV of cadmium atom became weak after the modification because PDbNbM was successfully covered by the surface of the QDs. PDbNbM bound on the QD nanoparticle was evaluated by hydrolysis of the poly(MEONP) segment. The concentration of released nitrophenoxy ion was determined by UV spectroscopy at 400 nm. The density of the PDbNbM chains on the QD was estimated roughly as 0.05 polymer chains/nm2. The most dense poly(MPC) brush reported is 0.39 polymer chains/cm2 36. That is at least 10 % of the surface area of the QD nanoparticle covered with the polymer. The phosphorylcholine group of the MPC polymer is bulky; thus, the surface of the QD was covered with the MPC polymer completely. The XPS analysis supported this speculation. The diameter of the obtained QD/PDbNbM nanoparticle in pure water was 39 ± 9 nm, and surface ζ-potential was 2.5 ± 0.4 mV. The diameter of the QD/PDbNbM nanoparticle changed in response to the pH value in the medium and it was reversible. The pH dependence of the diameter of the nanoparticle is indicated in Figure S5 in Supporting Information.

pH responsibility of QD/PDbNbM/Alexa nanoparticles The Alexa fluorescent dye was introduced to the PDbNbM chain in a reaction between active ester groups in the poly(MEONP) segment and the amino groups of Alexa. The fluorescence spectra of the QD/PDbNbM/Alexa nanoparticles were measured to


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understand the FRET phenomena relative to the medium pH. The emission wavelength of QD overlaps with the excitation wavelength of the Alexa dye; therefore, the energy transfer between QD and the Alexa dye is expected when they are in close proximity with each other (supporting information Figure S5). The FRET index is altered with changes in the conformation of the pH-dependent poly(DEAEMA) segment in the QD/PDbNbM/Alexa nanoparticles. We observed that the fluorescence property of both QD and Alexa dye was pH-insensitive in the pH range between 4.0 to 10.0. Figure 5 (a) shows the fluorescence spectra of the QD/PDbNbM/Alexa nanoparticles in aqueous medium with various pH values. A new fluorescence peak at 620 nm was detected in the fluorescence spectra measured at pH 7.4 and pH 9.0; however, the peak was not detected at pH 5.0. The FRET index shown in Figure 5 (b) was calculated from the fluorescence intensity. The FRET index was more enhanced at a higher pH, which was due to the conformational change in the pH-dependent poly(DEAEMA) segment. The poly(DEAEMA) pKa value is about 7.0; thus, protonation of the DEAEMA units induces stretch conformation of the poly(DEMEMA) segment at pH 5.0. On the other hand, the poly(DEMEMA) segment form shrinks when the pH is increased (pH responsible change in diameter of the QD/PDbNbM nanoparticle without Alexa dye is shown in Figure S6). This conformational change was accompanied by an altered distance between QD and the fluorescence dye. Additional triblock-type MPC polymers were synthesized to understand the effect of the distance between QD and Alexa on the FRET index in response to pH. Chemical structure and synthetic results are shown in the Supporting Information as Table S-1. It had longer poly(DEAEMA) segment (45 DEAEMA units) compared with the PDbNbM (22 DEMEMA units). We could not observe the FRET phenomenon even when the particle was in pH 9.0 conditions; this was due to the large distance between QD and Alexa. Moreover,


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we previously used poly[DEAEMA-block-(MPC-random-NEONP)], a diblock-type polymer, for modification of QD27. The QD/diblock-type polymer diameter was reduced when the pH was raised from 5.0 to 7.4. However, the FRET index did not increase significantly with the increase in pH. One explanation is the undefined position of Alexa in the polymer chain. Based on these results, we concluded that the triblock-type QD/PDbNbM/Alexa nanoparticles met the requirements to evaluate the pH in the cellular environment.

Cytocompatibility of R8-QD/PDbNbM/Alexa nanoparticles The CPP molecule must be used to enhance the internalization of the QD/PDbNbM/Alexa nanoparticles, because the nanoparticle covered with the MPC polymer is inert to cell membrane penetration26, 36. We selected R8 as the CPP and bound the end of the MPC polymer chains in a condensation reaction between a carboxyl and amino group in R8. The cytotoxicity of R8-mediated uptake of QD/PDbNbM/Alexa nanoparticle (R8QD/PDbNbM/Alexa nanoparticle) was evaluated in cultured HeLa cells, using the LDH assay. Figure 6 shows the relative cytotoxicity of PDbNbM and the nanoparticles. Cell cytotoxicity was lower after incubation with these polymers and nanoparticles compared to the Tween 20 control, even when they were at a high concentration. Previous studies reported that membrane damage is caused when LDH release is above 10 %, which was the value measured after incubation with the Triton-X control for 1 h37. Thus, cells incubated with the R8-QD/PDbNbM/Alexa nanoparticle did not have elevated cytotoxicity. We conclude that PDbNbM was beneficial for reduction of QD nanoparticles cytotoxicity.

Tracking location of the R8-QD/PDbNbM/Alexa nanoparticles in the cells and intracellular pH


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Figure 7 shows the fluorescence microscopy images of HeLa cells cultured with the R8-QD/PDbNbM/Alexa nanoparticles. Nanoparticles added to the cell culture medium began to interact with the cells. Initially, the fluorescence based on the nanoparticle was evenly distributed in the culture medium. The fluorescence signal was detected around the cells after 7 min of incubation, which became more apparent after 15 min. It reflected the accumulation of the nanoparticles around cells. Therefore, the R8-QD/PDbNbM/Alexa nanoparticles may interact with the cell membrane and have cellular uptake capability. HeLa cells were incubated with R8-QD/PDbNbM/Alexa for 150 min. The fluorescence generated by the nanoparticles was examined at 35 min, 75 min, and 120 min. Figure 8 shows CLSM images of HeLa cells after incubation with the R8QD/PDbNbM/Alexa nanoparticles. Hoechst-stained cell nuclei are shown in blue (Figure 8 (a)). Green fluorescence was clearly observed at 35–120 min of incubation, which was attributed to the QD. Red fluorescence was observed at 35 and 120 min of incubation, which corresponded to the emission wavelength of Alexa dye. The emission was carried out at a 473 nm light wavelength, which is the only wavelength that could excite QD. Thus, FRET was induced by QD and the Alexa dye because of the close proximity of these fluorescence probes to each other. However, the red fluorescence signal disappeared at the 75-min time point, and then was recovered at 120 min; and FRET was generated again. This corresponded to the shrink-stretch conformational change in the poly(DEAEMA) segment in response to the pH surrounding the nanoparticles. Many reports have described a pH change in the endosome during its transport process. Initially, the pH in the endosome is almost the same as the pH 7.4 outside the cell. After that, the endosome pH decreases to about 5.0, even though the pH in the cytoplasm is 7.4. It is known that the R8 induces transportation of compounds through cellular membrane by endocytosis

38, 39

. Therefore, our data indicate that the R8-

QD/PDbNbM/Alexa nanoparticles interacted with the cell membrane within 17 min, and


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were internalized through the cell membrane by endocytosis. Once the nanoparticles reached the endosome, the pH gradually decreased at 75 min. Then, the nanoparticles were transported from the endosome into the cytoplasm. The effect of export from the endosome is called the proton-sponge effect

40-42

. We successfully obtained continuous fluorescence

measurements during the transport of nanoparticles from outside of cell to internalization in the cell, transport inside the cell by endocytosis, and escape from the endosome. This was achieved by using cytocompatible and pH-response fluorescence nanoparticles. Figure 9 (a) shows a tracking trace of the R8-QD/PDbNbM/Alexa nanoparticle in the cell. The nanoparticle did not move significantly at 5 min of incubation. However, it moved into the cytoplasm after 100 min. The CLSM images enabled us to determine the nanoparticle velocity in the cell. The velocity of the nanoparticle corresponding to incubation time is shown in Figure 9 (b). The nanoparticles interacted with the cell membrane and initiated internalization within the first 20 min of incubation. Initially, the velocity of the nanoparticle was low, but it increased to more than 200 nm/min within 25 min. The velocities were 50– 150 nm/min during a 20–75 min incubation time. It suddenly increased to 250 nm/min at 75 min, and after that, it returned to 130 nm/min. The results in Figure 8 indicate that the nanoparticles were located in the endosome during the 20–75-min period. High velocity was observed when the nanoparticle was entrapped in the endosome and during their exit from the endosome. The continuous observation of the R8-QD/PDbNbM/Alexa nanoparticle movement provided a dynamic method for effectively tracking endocytosis of the nanoparticles. There are many low-molecular weight organic fluorescence dyes for measuring pH of the surrounding environment. These fluorescence dyes can also be used to measure the pH in cells. However, their ability to determine the local pH and time-dependent change in the pH is lost when the dye enters the organelles. Therefore, we think that the nanoparticle system


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based on the QD, such as the R8-QD/PDbNbM/Alexa nanoparticle, is superior compared with the organic fluorescence dye derivatives.

R8-QD/PDbNbM/Alexa nanoparticles entrapment in the endosome region The endosome entrapment of the R8-QD/PDbNbM/Alexa nanoparticles could be evaluated by FRET because the endosome region was detected by disappearance of energy transfer of the nanoparticles during a decrease in pH. The use of FRET made it possible to determine the number of endosomes in the cytosol. Figure 7 shows that the nanoparticles accumulated at the cell membrane surface were internalized by endocytosis; thus, the fluorescence from the QD in the cell corresponded to the number of endosomes. The QD/PDbNbM/Alexa nanoparticles could enter cells without cell-penetrating peptide R8. This was considered to be the effects of the cationic nature of the poly(DEAEMA) segment. When the number of nanoparticles in the feed was the same, the R8 could improve the celluptake rate and number of nanoparticles. Figure 10 (a) shows the number of endosomes that contained the R8QD/PDbNbM/Alexa nanoparticles during a 1-h incubation. The number of endosomes increased linearly with incubation time. Figure 10 (b) shows that the average size of an endosome that contained the nanoparticles was maintained in the range from 0.30 µm2 to 0.40 µm2. The size fluctuated in the first 20 min, and then became stable with no significant change in the size. These data indicate that the nanoparticles could be used to enhance our knowledge of the endocytosis process.

CONCLUSIONS This study introduced the well-defined triblock-type polymer, PDbNbM, for direct surface modification of QDs. The PDbNbM showed good pH-responsibility, which was


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attributed to protonation of the poly(DEAEMA) segment. The PDbNbM chains were able to bind to the hydrophobic layer of the QD nanoparticles by a solvent evaporation procedure. This modification enabled the QD/PDbNbM nanoparticles to be well dispersed in aqueous medium. FRET between QD and the fluorescence dye was observed under high-pH conditions after immobilization of the fluorescence dye to the poly(MEONP) segment. On the other hand, FRET disappeared under low-pH conditions because of a change in the poly(DEAEMA) segment conformation. Cellular internalization of the nanoparticles was enhanced by a cell-penetrating peptide, R8, immobilized at the end of the polymer chains. The R8-QD/PDbNbM/Alexa nanoparticles successfully detected intracellular movement, based on pH change during endocytosis, by their FRET phenomena. We conclude that QDs combined with cytocompatible MPC polymer chains provide a new imaging and transport nanodevice for cell-based science and engineering.

ASSOCIATED CONTENT Supporting Information This material is available free of charge on the ACS Publications website at DOI: Scheme of synthetic procedure of PDbNbM, NMR chart of PMPC, NMR chart of PNbM, NMR chart of PDbNbM, Chemical structure and synthetic results of additional triblock polymer PDbSbM, FRET phenomena between QD and fluorescence dye.

ACKNOWLEDGMENTS The authors thank Dr. Kyoko Fukazawa, The University of Tokyo, and Dr. Xiaojie Lin, University of Washington for their constructive suggestions. This research was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular


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Science” (No.2306), Ministry of Education, Culture, Sports, Science, and Technology of Japan.


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Figure 1. Chemical structure of triblock-type MPC polymer (PDbNbM). 549x380mm (72 x 72 DPI)


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Figure 2. Polymer concentration dependence on the surface tension of an aqueous solution. 549x380mm (72 x 72 DPI)


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Figure 3. Schematic representation of the structure of the QD/PDbNbM/Alexa nanoparticle. 549x380mm (72 x 72 DPI)


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Figure 4. XPS charts of QD and QD/PDbNbM nanoparticles. 549x380mm (72 x 72 DPI)


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Figure 5. Fluorescence spectra of the QD/PDbNbM/Alexa nanoparticles (a) and FRET index of the nanoparticle in various pH suspension (b). ((a): green line indicates fluorescence spectrum of QD; red line is Alexa; black line is QD/PDbNbM/Alexa at pH = 5.0; blue line is QD/PDbNbM/Alexa at pH = 7.4; yellow line is QD/PDbNbM/Alexa at pH = 9.0). 282x211mm (72 x 72 DPI)


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Figure 6. Relative cytotoxicity of polymer and nanoparticles determined by the LDH assay method. The LDH activity determined after addition of 0.5 wt% Tween 20 was defined as 100% injury rate. 549x380mm (72 x 72 DPI)


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Figure 7. Fluorescence microscopic image of R8-QD/PDbNbM/Alexa nanoparticles after addition of cell culture medium to HeLa cells at various incubation times. 549x380mm (72 x 72 DPI)


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Figure 8. CLSM image of R8-QD/PDbNbM/Alexa nanoparticle after incubation with HeLa cells. Blue fluorescence indicates nucleus stained by Hoechst dye, green fluorescence is attributed to QD, red fluorescence is attributed to Alexa. 549x380mm (72 x 72 DPI)


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Figure 9. Trace of R8-QD/PDbNbM/Alexa nanoparticle is indicated with blue line (a) and velocity of the nanoparticles in the cellular environment (b). 549x380mm (72 x 72 DPI)


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Figure 10. The number of endosomes containing R8-QD/PDbNbM/Alexa nanoparticles (a) and average area of endosome in the cells (b). 549x380mm (72 x 72 DPI)


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Table 1. Synthetic results of macroinitiators and triblock polymer, PDbNbM 282x211mm (72 x 72 DPI)


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Graphic abstract 549x412mm (72 x 72 DPI)


Movement of a Quantum Dot Covered with Cytocompatible and pH

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