Temperature-Induced Intracellular Uptake of ThermoresponsivePolymeric Micelles

Jun Akimoto,†,‡ Masamichi Nakayama,*,† Kiyotaka Sakai,‡ and Teruo Okano*,†

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns),Kawada-cho 8-1, Shinjuku, Tokyo 162-8666, Japan, and Department of Applied Chemistry, Waseda

University, Ohkubo 3-4-1, Shinjuku, Tokyo 169-8555, Japan

Received July 10, 2008; Revised Manuscript Received March 16, 2009

Well-defined diblock copolymers comprising thermoresponsive segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(IPAAm-co-DMAAm)) and hydrophobic segments of poly(D,L-lactide) were synthesizedby combination of RAFT and ring-opening polymerization methods. Terminal conversion of thermoresponsivesegments was achieved through reactions of maleimide or its Oregon Green 488 (OG) derivative with thiol groupsexposed by cleavage of polymer terminal dithiobenzoate groups. Thermoresponsive micelles obtained from thesepolymers were approximately 25 nm when below the lower critical solution temperature (LCST) of 40 °C, andtheir sizes increased to an average of approximately 600 nm above the LCST due to aggregation of the micelles.Interestingly, the OG-labeled thermoresponsive micelles showed thermally regulated internalization to culturedendothelial cells, unlike linear thermoresponsive P(IPAAm-co-DMAAm) chains. While intracellular uptake ofP(IPAAm-co-DMAAm) was extremely low at temperatures both below and above the micellar LCST, thethermoresponsive micelles showed time-dependent intracellular uptake above the LCST without exhibitingcytotoxicity. These results indicate that the new thermoresponsive micelle system may be a greatly promisingintracellular drug delivery tool.

Introduction

Poly(N-isopropylacrylamide) (PIPAAm) is well-known toexhibit a reversible temperature-responsive phase transitionthroughout its lower critical solution temperature (LCST) inaqueous media.1-3 This polymer is hydrophilic, existing in anextended conformation below its LCST, and undergoes a phasetransition to a water-insoluble, hydrophobic aggregate above32 °C. In addition, PIPAAm’s LCST can be easily controlledto near body temperature for biomedical applications byintroducing hydrophilic comonomers, such as N,N-dimethylacryl-amide.4,5 These unique features have been widely exploited toproduce materials with applications in the biomaterial, biosepa-ration, and drug delivery fields, including bioconjugates withenzymes6,7 or nucleic acids8,9 and controlled drug releasematrices.10-12 In our previous work, PIPAAm-grafted interfaceswere prepared to control interactions with bioactive componentssolely by using applied temperature changes. We successfullydemonstrated that the hydrophilic/hydrophobic switchable prop-erties of the grafts could be useful for various applicationsincluding aqueous chromatography systems to separate bioactivecompounds13-15 and new cell culture substrates for thermallycontrolled cell adhesion/detachment behavior.16-18 We and otherresearchers independently developed thermoresponsive poly-meric micelles comprising diblock copolymers of PIPAAmderivatives and various hydrophobic segments (e.g., poly(n-butylmethacrylate) and poly(D,L-lactide)) as systems to improvecancer chemotherapy.19-24 Multimolecular assemblies of blockor graft copolymers, polymeric micelles, are extremely attractivefor targeted drug delivery applications because of their unique

features such as reliable structural stability, nano-order diameter,and hydrophobic drug solubilization in aqueous milieu.25-27

These polymeric micelles escape from reticuloendothelialsystems (RES)28 and allow the accumulation of loaded drugspreferentially in solid tumor tissues through the enhancedpermeability and retention (EPR) effect.29,30 Our drug deliverystrategy using thermoresponsive micelle drug carrier systemsis a combination of conventional site-specific drug targeting withtemporal drug targeting modulated by local cancer therapeuticheating, hyperthermia.31

In our previous studies, several doxorubicin-loaded thermo-responsive micelles demonstrated successful controlled ON-OFFdrug release and subsequent controlled expression of in vitrocytotoxicity against endothelial or various cancer cells withapplied temperature changes.19-21,32 However, the influence ofthermally induced hydrophilic/hydrophobic property alternationson micellar interactions with cells and tissues remains incom-pletely understood. To further investigate micelle interactionswith cells and tissues, fluorescent labeling of polymeric micellesis effective for direct visualization of micellar localization.Recently, we successfully prepared surface-functionalized poly-meric micelles comprising well-defined end-functionalizedamphiphilic diblock copolymers using reversible addition-frag-mentation chain transfer (RAFT) radical polymerization.33,34 Inthe present report, we focus on thermally induced interactionswith cells and intracellular uptake by using fluorescent-labeledthermoresponsive micelles to explore possibilities for thermo-responsive micelles as intelligent drug carrier systems. For thispurpose, well-defined diblock copolymers of fluorescentlylabeled thermoresponsive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(IPAAm-co-DMAAm)) and poly(D,L-lactide) (PLA) were synthesized by combination of RAFTpolymerization35-37 and ring-opening polymerization. Chainterminal fluorescent probe introduction onto P(IPAAm-co-

* To whom correspondence should be addressed. Phone: +81-3-3353-8112, ext. 66201. Fax: +81-3358-7428. E-mail: tokano@abmes.twmu.ac.jp(T.O.); mnakayama@abmes.twmu.ac.jp (M.N.).

† Tokyo Women’s Medical University (TWIns).‡ Waseda University.

Biomacromolecules 2009, 10, 1331–1336 1331

10.1021/bm900032r CCC: $40.75 2009 American Chemical SocietyPublished on Web 04/09/2009

DMAAm) segments was achieved by reaction of maleimidederivatives with polymer thiol groups produced from aminolyzedterminal dithiobenzoate.33,38,39 The thermoresponsive polymericmicelles possessing fluorescent moieties on their outermostsurface were prepared through the assembly of end-function-alized diblock polymers into micelles (Figure 1)33 and werecharacterized for their hydrodynamic diameters and thermore-sponsive behavior. In addition, we further investigated theinfluence of temperature changes throughout the LCST on themicellar localization and uptake of cultured endothelial cellsby using confocal laser scanning microscopy and flow cytometry.

Experimental Section

Materials. N-Isopropylacrylamide (IPAAm) was kindly providedby Kojin (Tokyo, Japan) and purified by recrystallization from n-hexane.N,N-dimethylacrylamide (DMAAm, Wako Pure Chemicals, Osaka,Japan) was distilled under reduced pressure. 2,2′-Azobis[2-methyl-N-(hydroxyethyl)]propionamide (VA-086), 1,4-dioxane, tetrahydrofuran(THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc),xylene, and diethyl ether were purchased from Wako Pure Chemicals,and were used without further purification. D,L-Lactide (LA, TokyoChemical Industry, Tokyo, Japan) was recrystallized from ethyl acetate.2-Hydroxyethylamine (Kanto Chemical, Tokyo, Japan), maleimide(Mal, Aldrich, St. Louis, MO), tin(II) 2-ethylhexanoate (Aldrich), andOregon Green 488 maleimide (Invitrogen, Carlsbad, CA) were usedas received. A RAFT agent, 2-[N-(2-hydroxyethyl)carbamoyl]prop-2-yl dithiobenzoate (HECPD) was prepared according to a previouslypublished procedure.40

Preparation of Diblock Copolymers.41R-Hydroxyl, ω-thiobenzoyl-thio-P(IPAAm-co-DMAAm) (TBT-P(IPAAm-co-DMAAm)-OH) wassynthesized in 1,4-dioxane, employing HECPD (30 mM) and VA-086(6 mM) as the RAFT agent and initiator, respectively. Initial totalmonomer concentration was 3 M (IPAAm/DMAAm ) 70/30 mol %).The polymerization was conducted at 85 °C for 25 h after degasificationthrough three freeze-pump-thaw cycles of the monomer solution.Polymers were purified by repeated precipitation in excess diethyl ether,followed by drying under vacuum. In the next step of diblock copolymersynthesis, second block was prepared by ring-opening polymerizationof LA (6.5 × 10-3 mol) in xylene using R-hydroxylated P(IPAAm-co-DMAAm) (5.4 × 10-5 mol) and tin(II) 2-ethylhexanoate asmacroinitiator and catalyst, respectively. Polymerization was performedat 110 °C for 12 h in a nitrogen atmosphere. Polymers were precipitated

in excess diethyl ether and then dried in vacuo. The obtained polymerswere characterized by using 1H NMR (400 MHz, Varian, CA) and gelpermeation chromatography (GPC). GPC analysis was performed ona GPC system (SC-8020, Tosoh, Tokyo, Japan) with two columns(TSKgel-G3000H HR and TSKgel-G4000H HR, Tosoh) at 45 °C usingDMF containing 100 mM LiCl as eluent (elution rate, 1.0 mL/min)and polyethylene oxide standards.

Aminolysis and Conversion of Polymer Terminal Groups.41Theobtained polymers (150 mg) were dissolved in 8 mL of deoxidizedTHF, including either maleimide (40 mol equiv vs polymer termini)or Oregon Green 488 maleimide (10 mol equiv vs polymer termini).2-Hydroxyethylamine (10 mol equiv vs polymer termini) was addedto polymers solutions, and then reactions were carried out at 25 °C for20 h in a nitrogen atmosphere under dark condition. After reaction,polymer solutions were dialyzed against pure water until completeremoval of unreacted maleimide compounds. Polymers were recoveredby lyophilization as powder.

Preparation and Characterization of Polymeric Micelles. Diblockcopolymers of maleimide (Mal)-terminated P(IPAAm-co-DMAAm)-b-PLA were dissolved in DMAc, and solutions were then dialyzedagainst pure water using dialysis membranes (Spectra/Por 6, MWCO1000, Spectrum Laboratories, CA) at 10 °C for 24 h. The obtainedmicelles were coded as M(Mal). Oregon Green 488 (OG)-labeledmicelles (M(OG)) were prepared by the same procedure using polymermixtures of Mal- and OG-P(IPAAm-co-DMAAm)-b-PLA (Mal/OG )80/20 in polymer wt %) under dark condition. Hydrodynamic micellardiameters and their distributions in Dulbecco’s phosphate buffered salinewithout calcium chloride and magnesium chloride (DPBS(-), pH7.4,Sigma, St. Louis, MO) were determined by dynamic light scattering(DLS) using a DLS-7000 instrument (Otsuka Electronics, Tokyo, Japan)with a He-Ne laser (633 nm) at a scattering angle of 90°. Opticaltransmittance of the polymeric micelles (5.0 mg/ml) in DPBS(-) atvarious temperatures were measured at 600 nm by UV-vis spectro-photometry (V-530, JASCO, Tokyo, Japan) with a sample cellthermostat (EHC-477S, JASCO). Heating rate was 0.1 °C/min. TheLCSTs of the micelle solutions were defined as the temperatureproducing a 50% decrease in optical transmittance.

Cell Culture. Bovine carotid endothelial cells (EC, Health ScienceResearch Resources Bank, Osaka, Japan) were cultured in Dulbecco’smodified Eagle’s medium (DMEM, Sigma) supplemented with 10%fetal bovine serum (FBS, Bioserum, Victoria, Australia), 50 units/mLpenicillin, and 50 µg/mL streptomycin at 37 °C under 5% CO2

Figure 1. (a) Synthesis of P(IPAAm-co-DMAAm)-b-PLA diblock copolymers. (b) Conversion of thermoresponsive polymer termini and formationof polymeric micelles.

1332 Biomacromolecules, Vol. 10, No. 6, 2009 Akimoto et al.

condition. Cells were cultured for 2 days to achieve approximatelyconfluent conditions before performing all experiments.

Confocal Laser Scanning Microscopy (CLSM). ECs were seededinto 4-well Lab-Tek chambered cover glasses (2.0 × 105 cells/mL, 500µL/well, Nalge Nunc International, Rochester, NY) and then culturedfor 2 days. Cultured cells were exposed to OG-labeled micelles (200µg/mL) in DMEM/FBS at the temperatures below (37 °C) or above(42 °C) the LCST for 9 h in a humidified atmosphere with 5% CO2.After incubation, ECs were rinsed with DMEM/FBS and incubatedfor another 30 min with Cell Tracker Red (Invitrogen, 10 µM inDMEM) at 37 °C. ECs were fixed with 4% paraformaldehyde (WakoPure Chemicals) in DPBS(-) for 5 min, rinsed with DPBS(-) twice.Cell nuclei were stained with Hoechst 33258 (Invitrogen) for 5 min,followed by two rinses with DPBS(-). Samples were visualized by TCSSP confocal laser scanning microscope (Leica, Germany) with Ar/Krand Ar/UV lasers.

Flow Cytometry. ECs were seeded into 4-well multidishes (2.0 ×105 cells/mL, 500 µL/well, Nalge Nunc International), followed by a 2day cultivation. ECs were incubated with polymer micelles (200 µg/mL M(OG)) in DMEM/FBS at the temperatures below (37 °C) or above(42 °C) the LCST. After incubation for various periods, ECs were gentlyrinsed with DMEM/FBS to remove nonintracellular OG-labeled mi-celles, followed by treatment with 0.05% trypsin-EDTA (Sigma).Recovered cells were rinsed with DPBS(-) twice and 1 µL of propidiumiodide (1 mg/mL, Invitrogen) was added to sample solutions. Cellularuptake was estimated using an EPICS XL-MCL flow cytometer(Beckman coulter, Fullerton, CA) and 10000 events were analyzed usingEXPO2 software.

Cytotoxity Assays of Polymeric Micelles. ECs (2.0 × 105 cells/mL, 100 µL/well) were seeded into 96-well microplates (Falcon 3072,BD Biosciences, Franklin Lakes, NJ) and then cultured for 2 days.The cells were incubated with polymeric micelles at various concentra-tions (0.01-1 mg/mL M(Mal)) in DMEM/FBS at temperaturesthroughout the LCST (37 or 42 °C) for 24 h. After incubation withmicelles, ECs were gently rinsed with DMEM/FBS twice. The mediawere replaced with 100 µL of Cell Counting Kit-8 (DOJINDO,Kumamoto, Japan), followed by incubation under 5% CO2 at 37 °Cfor 2.5 h. Then absorbance was measured at 450 nm using a microplatespectrophotometer (SPECTRAmax250, Molecular Devices, Sunnyvale,CA). Surviving cells were calculated according to the followingequation:

cell viability(%) ) (ODsample - ODblank)/

(ODcontrol - ODblank) × 100

where ODsample represents absorbance of test well, ODcontrol representsabsorbance of positive growth control well (incubated without micellesat 37 °C), and ODblank represents absorbance of only Cell CountingKit-8.

Results and Discussion

Preparation of Diblock Copolymers. ΤΒΤ-P(IPAAm-co-DMAAm)-OH was obtained as pinkish white powder by RAFTpolymerization using HEPCD as the hydroxyl RAFT agent(yield, 62%). The obtained thermoresponsive polymer wasdetermined to be nearly monodisperse (polydispersity index, PDI) 1.08) by GPC (Table 1). The 1H NMR spectrum inchloroform-d (CDCl3) showed three peaks derived from theterminal dithiobenzoate group at 7.35, 7.55, and 7.95 ppm,corresponding to signals of meta-, para-, and ortho-phenyl,respectively (see Figure S1 in Supporting Information). Number-averaged molecular weight (Mn) and chemical composition ofP(IPAAm-co-DMAAm) were determined by 1H NMR, estimat-ing from the integrated proton signals derived from IPAAmmethine (4.00 ppm), DMAAm methyl (2.90 ppm), and ortho-phenyl of terminal dithiobenzoate (7.95 ppm; Mn ) 9300,

monomer unit: IPAAm/DMAAm ) 54/29). DMAAm composi-tion in the thermoresponsive polymers was approximately 5 mol% higher than initial feed DMAAm composition, and monomerratio was controlled by the initial monomer composition.33 Thepolymers in DMF showed a n-π* absorption band (λmax ) 498nm) corresponding to terminal dithiobenzoate groups, and Mn

by end group analysis was 9600, calculated from the molarextinction coefficient of terminal dithiobenzoate (determined as109.5 L mol-1 cm-1 in DMF). In addition, Mn calculated fromGPC was 10000, in good agreement with the results of polymerend group assays by both 1H NMR and UV spectrometricmeasurements. In the second step of the polymer synthesis,diblock copolymers with biodegradable segments were preparedby ring-opening polymerization of LA initiated by P(IPAAm-co-DMAAm) R-hydroxyl groups using tin(II) 2-ethylhexanoateas the catalyst. ω-Dithiobenzoate groups in RAFT polymersthermally decompose at a temperature over 120 °C.42 Therefore,we optimized the polymerization condition (110 °C, 12 h) andsuccessfully obtained diblock polymers with a high ω-func-tionality (93%, determined by 1H NMR). The resultant diblockcopolymers had a low PDI of 1.07 by GPC. According to 1HNMR studies of the diblock copolymers, Mn and monomercompositions were then determined by the peak areas of IPAAmmethine (4.00 ppm), DMAAm methyl (2.90 ppm), and LAmethylene (5.20 ppm) protons. Additionally, Mn obtained fromGPC was 12000, comparable to the molecular weight deter-mined by 1H NMR. The characterization of these polymers issummarized inTable 1.

Aminolysis and Conversion of Polymer Termini. Animportant feature specific to RAFT polymerization is thepossibility to prepare polymers possessing terminal dithioestergroups that can be easily aminolyzed with primary or secondaryamines, producing terminal thiol groups43,44 (Figure 1b). Thisfeature of RAFT technique has potential for molecular designand preparation of functional polymers including bioconjugatedpolymers and fluorescently labeled polymers by thiol couplingchemistry.33,38,45 In this work, we attempted substitutions ofthe corona-forming thermoresponsive polymer termini by afacile maleimide-thiol coupling reaction. TBT-P(IPAAm-co-DMAAm)-b-PLA diblock polymers were pink in color due toa n-π* absorption band around 500 nm corresponding to theω-dithiobenzoate groups. After polymer terminal aminolysis,the absorption completely disappeared. We adopted a one-potreaction of both ω-dithiobenzoate aminolysis and couplingreaction with maleimide derivatives for highly efficient terminalsubstitution. GPC elution curves and PDI values of diblockcopolymers were comparable before and after the terminalcoupling reaction, polymers reacted with maleimide, and its OG-derivative showed narrow PDIs of 1.06 and 1.11, respectively.

Formation and Characterization of Polymeric Micelles.Core-corona type thermoresponsive polymeric micelles wereobtained by dialysis of P(IPAAm-co-DMAAm)/PLA blockcopolymers in DMAc against water at 10 °C.46 We havepreviously reported that surface functional groups influenced

Table 1. Characterization of Polymer Compositions andMolecular Weights

polymer sample codemonomer unitsa

IPAAm/DMAAm/LA Mnb PDIc

TBT-P(IPAAm-co-DMAAm)-OH 54/29/- 9300 1.08TBT-P(IPAAm-co-DMAAm)-b-PLA 54/29/14 11300 1.07

a Estimated by 1H NMR. b Number-averaged molecular weight, Mn,estimated by 1H NMR. c Determined by GPC using DMF with 100 mMLiCl.

Intracellular Uptake of Polymeric Micelles Biomacromolecules, Vol. 10, No. 6, 2009 1333

on the micellar LCST.33 In addition, we consider that highlyconcentrated fluorescent moieties on the micellar surfaces maylead to fluorescent quenching. Therefore, in this work, OG-labeled polymeric micelles were prepared by mixing of Mal-and OG-P(IPAAm-co-DMAAm)-b-PLA (Mal/OG ) 80/20 inpolymer wt %). Obtained P(IPAAm-co-DMAAm)/PLA micellesolutions were highly transparent and shown to have nanoscalediameters and monomodal size distribution at 37 °C, regardlessof surface functional group differences (Table 2). In addition,the polymeric micelles showed a monomodal size distributionin the presence of bovine serum albumin (see Figure S5 inSupporting Information), suggesting limited interaction withserum proteins by densely packed thermoresponsive polymerchains of the micelle outer shells.

For passive drug targeting using carrier systems, nano-ordercarrier sizes (ca. 5-200 nm) are strongly desirable for long-term circulation in the bloodstream, avoiding both renal filtrationand RES uptake,28 and for subsequent selective tumor ac-cumulation based on specific macroscopic properties of solidtumors, the EPR effect.29,30 We investigated the micellarthermoresponsive behavior in DPBS(-) by measuring opticaltransmittance changes at various temperatures with results shownin Table 2. P(IPAAm-co-DMAAm)/PLA micelles exhibited aphase transition throughout the LCSTs (ca. 40 °C) based onhydrophilic/hydrophobic switching of the corona-forming ther-moresponsive polymer segments. The obtained thermorespon-sive polymeric micelles can be utilized as the intelligent drugcarrier in conjunction with local heating cancer therapy at 42°C.31 Upon heating micellar solutions of M(Mal) and M(OG)at the temperature above the LCST (42 °C), the hydrodynamicdiameters increased and formed unimodal submicron-orderedaggregates of 614 and 589 nm, respectively (Table 2 and Figure2). The polymer aggregation was caused by the alternation ofthe micellar corona property from hydrophilic to hydrophobic,and subsequent promotion of hydrophobic interaction betweenthermoresponsive chains in aqueous media. The introductionof OG moieties to the micellar surfaces scarcely affected the

micellar properties including size distribution and thermore-sponsive behavior.

Cellular Uptake and Intracellular Distribution of thePolymeric Micelles. Effects of differences in temperaturethroughout the LCST on intracellular uptake of polymer micelleswere investigated by confocal laser scanning microscopy(CLSM) using OG-labeled thermoresponsive micelles (200 µg/mL, LCST ) 39.4 °C, critical micelle concentration) 22 µg/mL41). We confirmed that OG maleimide derivatives did notshow any cytotoxicity and internalize to the cultured cells (datanot shown), and considered that influence of OG introductionto the micellar surfaces on cytotoxicity and cellular uptake wasnegligible. Before the microscopy experiments, the cells incu-bated with the micelles were gently and thoroughly rinsed withDPBS(-) to remove any micelles adhered to the cell surfaces at25 °C. In the CLSM images, the green fluorescence derivedfrom the OG-labeled micelles was negligible below the micelleLCST (37 °C, Figure 3a). Of great interest, the fluorescencefrom micelles inside the cells was clearly observed above theLCST (42 °C), and green-colored dots were localized aroundthe cell nuclei (Figure 3b). We have obtained similar CLSMimages of the micelle-treated cells without cytological fixation,indicating that fixation treatments did not affect on the perme-ability of cellular membranes. We further investigated timedependence and efficiency of temperature-modulated intracel-lular uptake by flow cytometry. According to the flow cytometricstudies, cellular uptake of the micelles incubated at 37 °C (belowthe LCST) was considerably low after 24 h (Figure 4). Bycontrast, the micelles incubated at 42 °C (above the LCST) wereclearly detected within the cells at 2 h and later. The intensityof OG fluorescence derived from these micelles kept increasingup to 6 h, to approximately 16-fold greater intracellular uptake(Figure 4a). Additionally, we investigated effects of temperaturechanges on cellular membrane permeability using propidiumiodide. Cellular membrane permeability was independent ofincubation temperatures ranging from 37 to 42 °C, indicatingthat cellular uptake of the micelles did not result from changesof membrane permeability at elevated temperature. We furtherinvestigated influence of FBS on intercellular uptake of thepolymeric micelles. Relative cellular uptake of the micelleswithout FBS was 74.5 ( 1.1% (n ) 4) compared to that withFBS. In the DLS studies, the micelles did not significantlyinteract with the serum albumins at temperatures both belowand above the LCST (see Figure S5 in Supporting Information).Therefore, the decrease in cellular uptake of the micelles wasprobably caused by lowered cell growth and viability in absenceof FBS. We further investigated effects of the polymer assemblyon thermally modulated intracellular localization using OG-labeled P(IPAAm-co-DMAAm) (Mn ) 10300, Mw/Mn ) 1.07,200 µg/mL). As shown in Figure 4b, cellular uptake of the OG-labeled P(IPAAm-co-DMAAm) was comparable to that of thethermoresponsive micelles at 37 °C (below LCST). In addition,cellular uptake of the thermoresponsive linear polymers wasnot affected by temperature change throughout the LCST from37 to 42 °C. No temperature effect on cellular uptake of linear

Table 2. Characterization of OG-Labeled Thermoresponsive Polymers and Polymeric Micelles

37 °C 42 °C

sample code LCSTb (°C) diameter (nm) PDI diameter (nm) PDI

P(IPAAm-co-DMAAm) 40.2 8.1 ( 1.6 0.14 8.4 ( 1.8 0.22M(Mal) 40.0 23.3 ( 12.8 NDc 613.9 ( 273.6 NDc

M(OG)a 39.4 20.7 ( 8.5 0.20 588.8 ( 73.3 0.041a Micelles comprising mixtures of Mal- and OG-terminated block copolymers at the ratio 80/20 (wt %). b Determined by optical transmittance changes

in DPBS(-). c Not determined.

Figure 2. Size distribution of OG-labeled thermoresponsive polymericmicelles at temperatures below (37 °C, open circle) and above (42°C, closed circle) the LCST in DPBS(-).

1334 Biomacromolecules, Vol. 10, No. 6, 2009 Akimoto et al.

polymers was observed because dehydrated polymer chains didnot aggregate at the polymer concentration of 0.2 mg/mL abovethe LCST as shown in Table 2. The enhancement of intracellularuptake for the polymeric micelle system seems to be due totwo possible mechanisms. First, hydrophobic interactionsbetween the cell membranes and hydrophobic micelle cores werepromoted by collapse of the thermoresponsive corona-formingpolymers above the LCST. Below the LCST, densely packedand hydrated thermoresponsive corona-forming P(IPAAm-co-DMAAm) reduced possible interactions of the PLA cores withcell surfaces. Upon temperature increase above the LCST, thecorona-forming polymer chains collapse with the dehydrationof IPAAm units. Because of significant polymer conformationalchanges, hydrophobic interactions between the micelles and cellmembranes increase. Consequently, adhesion of the micelles

to the cell surfaces was promoted, followed by enhancementof intracellular uptake. A second possibility is that the coronaPIPAAm derivatives attached to the hydrophobic polymericcores regulate micelle adhesion to cell surfaces and sustainedintracellular uptake. We have previously reported that ultrathinPIPAAm-grafted layers (<20 nm) on polystyrene surfacessuccessfully achieved temperature-modulated cell adhesion/detachment.47 This is due to progressive dehydration andaggregation of PIPAAm segments at the hydrophobic polysty-rene interface with reduced molecular motion of covalentlybound PIPAAm chains. Thus, ultrathin PIPAAm layers onhydrophobic substrates demonstrate greater hydrophobic proper-ties than thicker polymer layers and play a critical role in celladhesion/detachment behavior.

In addition, we investigated polymer cytotoxicity (0.01-1mg/mL M(Mal)) by determining the viabilities of ECs at culturetemperatures below and above the micellar LCST (Figure 5).The viabilities of ECs incubated with micelles were at the samelevel as those ECs without exposure to the micelles at both 37and 42 °C, regardless of any increase in polymer concentration.Lower cell viabilities at 42 °C were observed compared withthose at 37 °C, due to only the influence of elevated temperature;the viability of only thermally treated ECs without the micellesalso demonstrated a lower value of 97.7 ( 1.7%. These resultsindicate that the polymer cytotoxicity of P(IPAAm-co-DMAAm)/PLA micelles is negligible, showing that micelles prepared inthis work are biocompatible materials. Consequently, thethermoresponsive micelle systems can be exploited as intelligentdrug targeting tools by combinations with local heating sys-tems.31

Figure 3. CLSM images of polymeric micelles localized within cultured cells after incubation for 9 h (a) below the LCST (37 °C) and (b) abovethe LCST (42 °C) in 10% serum culture media. The nuclei and cytoplasm were stained with Hoechst 33258 (blue) and Cell Tracker Red (red),respectively. Green fluorescence was derived from OG-labeled micelles. Scale bars: 50 µm.

Figure 4. Time-dependent cellular uptake of (a) OG-labeled thermo-responsive micelles and (b) the OG-labeled P(IPAAm-co-DMAAm)at temperatures below (37 °C, open circle) and above (42 °C, closedcircle) their LCST (mean ( SD, n ) 4). Y-axis: mean fluorescentintensity of 10000 events.

Figure 5. Viabilities of cultured endothelial cells incubated with Mal-labeled polymer micelles at various concentrations at 37 °C (opencircle) and above 42 °C (closed circle; mean ( S.D., n ) 8).Incubation time: 24 h.

Intracellular Uptake of Polymeric Micelles Biomacromolecules, Vol. 10, No. 6, 2009 1335

Conclusions

In summary, surface fluorescently labeled thermoresponsivepolymeric micelles were prepared using well-defined end-functionalized diblock copolymers for investigation of theirtemperature-modulated interactions with cultured cells by con-focal laser scanning microscopy and flow cytometry. Wesuccessfully demonstrated specific temperature effects promotingmicelle localization within cultured cells in the presence ofserum. Intracellular uptake was significantly increased at atemperature only above the micelle LCST due to the enhance-ment of interactions between cells and micelles mediatedthrough the thermoresponsive phase transition of the micellecoronas. In addition, the P(IPAAm-co-DMAAm)/PLA micellesshowed negligible cytotoxity to the cultured cells, independentof temperature changes. This specific temperature-controlledcellular uptake system using thermoresponsive polymeric mi-celles can be useful as intracellular delivery systems foranticancer drugs, genes, and peptides by combination withclinically applied heating systems.

Acknowledgment. The authors are grateful to Prof. D. W.Grainger (University of Utah) for his valuable comments andmanuscript editing. NEDO Special Courses for Developmentof Innovative Drug Delivery Systems funded by New Energyand Industrial Technology Development Organization (NEDO),Japan, Global COE program “Center for Practical ChemicalWisdom” supported by Ministry of Education, Culture, Sports,Science and Technology (MEXT), and the Grant-in-Aid forYoung Scientists (B; No. 19700407) from Japan Society forPromotion of Science (JSPS) are also acknowledged.

Supporting Information Available. 1H NMR, GPC, and UVdata of the polymers, and fluorescent, DLS, and flow cytometrydata of the micelles. This material is available free of chargevia the Internet at http://pubs.acs.org.

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