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Temperature-Induced Intracellular Uptake of Thermoresponsive Polymeric 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,Ndimethylacrylamide) (P(IPAAm-co-DMAAm)) and hydrophobic segments of poly(D,L-lactide) were synthesized by combination of RAFT and ring-opening polymerization methods. Terminal conversion of thermoresponsive segments was achieved through reactions of maleimide or its Oregon Green 488 (OG) derivative with thiol groups exposed by cleavage of polymer terminal dithiobenzoate groups. Thermoresponsive micelles obtained from these polymers were approximately 25 nm when below the lower critical solution temperature (LCST) of 40 °C, and their 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 cultured endothelial cells, unlike linear thermoresponsive P(IPAAm-co-DMAAm) chains. While intracellular uptake of P(IPAAm-co-DMAAm) was extremely low at temperatures both below and above the micellar LCST, the thermoresponsive micelles showed time-dependent intracellular uptake above the LCST without exhibiting cytotoxicity. These results indicate that the new thermoresponsive micelle system may be a greatly promising intracellular drug delivery tool.
Introduction Poly(N-isopropylacrylamide) (PIPAAm) is well-known to exhibit a reversible temperature-responsive phase transition throughout its lower critical solution temperature (LCST) in aqueous media.1-3 This polymer is hydrophilic, existing in an extended conformation below its LCST, and undergoes a phase transition to a water-insoluble, hydrophobic aggregate above 32 °C. In addition, PIPAAm’s LCST can be easily controlled to near body temperature for biomedical applications by introducing hydrophilic comonomers, such as N,N-dimethylacrylamide.4,5 These unique features have been widely exploited to produce materials with applications in the biomaterial, bioseparation, and drug delivery fields, including bioconjugates with enzymes6,7 or nucleic acids8,9 and controlled drug release matrices.10-12 In our previous work, PIPAAm-grafted interfaces were prepared to control interactions with bioactive components solely by using applied temperature changes. We successfully demonstrated that the hydrophilic/hydrophobic switchable properties of the grafts could be useful for various applications including aqueous chromatography systems to separate bioactive compounds13-15 and new cell culture substrates for thermally controlled cell adhesion/detachment behavior.16-18 We and other researchers independently developed thermoresponsive polymeric micelles comprising diblock copolymers of PIPAAm derivatives and various hydrophobic segments (e.g., poly(n-butyl methacrylate) and poly(D,L-lactide)) as systems to improve cancer chemotherapy.19-24 Multimolecular assemblies of block or graft copolymers, polymeric micelles, are extremely attractive for targeted drug delivery applications because of their unique * To whom correspondence should be addressed. Phone: +81-3-33538112, ext. 66201. Fax: +81-3358-7428. E-mail:
[email protected] (T.O.);
[email protected] (M.N.). † Tokyo Women’s Medical University (TWIns). ‡ Waseda University.
features such as reliable structural stability, nano-order diameter, and hydrophobic drug solubilization in aqueous milieu.25-27 These polymeric micelles escape from reticuloendothelial systems (RES)28 and allow the accumulation of loaded drugs preferentially in solid tumor tissues through the enhanced permeability and retention (EPR) effect.29,30 Our drug delivery strategy using thermoresponsive micelle drug carrier systems is a combination of conventional site-specific drug targeting with temporal drug targeting modulated by local cancer therapeutic heating, hyperthermia.31 In our previous studies, several doxorubicin-loaded thermoresponsive micelles demonstrated successful controlled ON-OFF drug release and subsequent controlled expression of in vitro cytotoxicity against endothelial or various cancer cells with applied temperature changes.19-21,32 However, the influence of thermally induced hydrophilic/hydrophobic property alternations on micellar interactions with cells and tissues remains incompletely understood. To further investigate micelle interactions with cells and tissues, fluorescent labeling of polymeric micelles is effective for direct visualization of micellar localization. Recently, we successfully prepared surface-functionalized polymeric micelles comprising well-defined end-functionalized amphiphilic diblock copolymers using reversible addition-fragmentation chain transfer (RAFT) radical polymerization.33,34 In the present report, we focus on thermally induced interactions with cells and intracellular uptake by using fluorescent-labeled thermoresponsive micelles to explore possibilities for thermoresponsive micelles as intelligent drug carrier systems. For this purpose, well-defined diblock copolymers of fluorescently labeled thermoresponsive poly(N-isopropylacrylamide-co-N,Ndimethylacrylamide) (P(IPAAm-co-DMAAm)) and poly(D,Llactide) (PLA) were synthesized by combination of RAFT polymerization35-37 and ring-opening polymerization. Chain terminal fluorescent probe introduction onto P(IPAAm-co-
10.1021/bm900032r CCC: $40.75 2009 American Chemical Society Published on Web 04/09/2009
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Figure 1. (a) Synthesis of P(IPAAm-co-DMAAm)-b-PLA diblock copolymers. (b) Conversion of thermoresponsive polymer termini and formation of polymeric micelles.
DMAAm) segments was achieved by reaction of maleimide derivatives with polymer thiol groups produced from aminolyzed terminal dithiobenzoate.33,38,39 The thermoresponsive polymeric micelles possessing fluorescent moieties on their outermost surface were prepared through the assembly of end-functionalized diblock polymers into micelles (Figure 1)33 and were characterized for their hydrodynamic diameters and thermoresponsive behavior. In addition, we further investigated the influence of temperature changes throughout the LCST on the micellar localization and uptake of cultured endothelial cells by using confocal laser scanning microscopy and flow cytometry.
Experimental Section Materials. N-Isopropylacrylamide (IPAAm) was kindly provided by 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, Tokyo Chemical 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), and Oregon Green 488 maleimide (Invitrogen, Carlsbad, CA) were used as received. A RAFT agent, 2-[N-(2-hydroxyethyl)carbamoyl]prop-2yl dithiobenzoate (HECPD) was prepared according to a previously published procedure.40 Preparation of Diblock Copolymers.41R-Hydroxyl, ω-thiobenzoylthio-P(IPAAm-co-DMAAm) (TBT-P(IPAAm-co-DMAAm)-OH) was synthesized in 1,4-dioxane, employing HECPD (30 mM) and VA-086 (6 mM) as the RAFT agent and initiator, respectively. Initial total monomer concentration was 3 M (IPAAm/DMAAm ) 70/30 mol %). The polymerization was conducted at 85 °C for 25 h after degasification through 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 copolymer synthesis, second block was prepared by ring-opening polymerization of LA (6.5 × 10-3 mol) in xylene using R-hydroxylated P(IPAAmco-DMAAm) (5.4 × 10-5 mol) and tin(II) 2-ethylhexanoate as macroinitiator and catalyst, respectively. Polymerization was performed at 110 °C for 12 h in a nitrogen atmosphere. Polymers were precipitated
in excess diethyl ether and then dried in vacuo. The obtained polymers were characterized by using 1H NMR (400 MHz, Varian, CA) and gel permeation chromatography (GPC). GPC analysis was performed on a GPC system (SC-8020, Tosoh, Tokyo, Japan) with two columns (TSKgel-G3000H HR and TSKgel-G4000H HR, Tosoh) at 45 °C using DMF containing 100 mM LiCl as eluent (elution rate, 1.0 mL/min) and polyethylene oxide standards. Aminolysis and Conversion of Polymer Terminal Groups. 41The obtained polymers (150 mg) were dissolved in 8 mL of deoxidized THF, 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 added to polymers solutions, and then reactions were carried out at 25 °C for 20 h in a nitrogen atmosphere under dark condition. After reaction, polymer solutions were dialyzed against pure water until complete removal of unreacted maleimide compounds. Polymers were recovered by lyophilization as powder. Preparation and Characterization of Polymeric Micelles. Diblock copolymers of maleimide (Mal)-terminated P(IPAAm-co-DMAAm)b-PLA were dissolved in DMAc, and solutions were then dialyzed against pure water using dialysis membranes (Spectra/Por 6, MWCO 1000, Spectrum Laboratories, CA) at 10 °C for 24 h. The obtained micelles were coded as M(Mal). Oregon Green 488 (OG)-labeled micelles (M(OG)) were prepared by the same procedure using polymer mixtures of Mal- and OG-P(IPAAm-co-DMAAm)-b-PLA (Mal/OG ) 80/20 in polymer wt %) under dark condition. Hydrodynamic micellar diameters and their distributions in Dulbecco’s phosphate buffered saline without 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°. Optical transmittance of the polymeric micelles (5.0 mg/ml) in DPBS(-) at various temperatures were measured at 600 nm by UV-vis spectrophotometry (V-530, JASCO, Tokyo, Japan) with a sample cell thermostat (EHC-477S, JASCO). Heating rate was 0.1 °C/min. The LCSTs of the micelle solutions were defined as the temperature producing a 50% decrease in optical transmittance. Cell Culture. Bovine carotid endothelial cells (EC, Health Science Research Resources Bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Bioserum, Victoria, Australia), 50 units/mL penicillin, and 50 µg/mL streptomycin at 37 °C under 5% CO2
Intracellular Uptake of Polymeric Micelles condition. Cells were cultured for 2 days to achieve approximately confluent conditions before performing all experiments. Confocal Laser Scanning Microscopy (CLSM). ECs were seeded into 4-well Lab-Tek chambered cover glasses (2.0 × 105 cells/mL, 500 µL/well, Nalge Nunc International, Rochester, NY) and then cultured for 2 days. Cultured cells were exposed to OG-labeled micelles (200 µg/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 incubated for another 30 min with Cell Tracker Red (Invitrogen, 10 µM in DMEM) at 37 °C. ECs were fixed with 4% paraformaldehyde (Wako Pure 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 TCS SP confocal laser scanning microscope (Leica, Germany) with Ar/Kr and 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 2 day 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 gently rinsed with DMEM/FBS to remove nonintracellular OG-labeled micelles, followed by treatment with 0.05% trypsin-EDTA (Sigma). Recovered cells were rinsed with DPBS(-) twice and 1 µL of propidium iodide (1 mg/mL, Invitrogen) was added to sample solutions. Cellular uptake was estimated using an EPICS XL-MCL flow cytometer (Beckman coulter, Fullerton, CA) and 10000 events were analyzed using EXPO2 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 concentrations (0.01-1 mg/mL M(Mal)) in DMEM/FBS at temperatures throughout the LCST (37 or 42 °C) for 24 h. After incubation with micelles, ECs were gently rinsed with DMEM/FBS twice. The media were replaced with 100 µL of Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan), followed by incubation under 5% CO2 at 37 °C for 2.5 h. Then absorbance was measured at 450 nm using a microplate spectrophotometer (SPECTRAmax250, Molecular Devices, Sunnyvale, CA). Surviving cells were calculated according to the following equation:
cell viability(%) ) (ODsample - ODblank)/ (ODcontrol - ODblank) × 100 where ODsample represents absorbance of test well, ODcontrol represents absorbance of positive growth control well (incubated without micelles at 37 °C), and ODblank represents absorbance of only Cell Counting Kit-8.
Results and Discussion Preparation of Diblock Copolymers. ΤΒΤ-P(IPAAm-coDMAAm)-OH was obtained as pinkish white powder by RAFT polymerization using HEPCD as the hydroxyl RAFT agent (yield, 62%). The obtained thermoresponsive polymer was determined to be nearly monodisperse (polydispersity index, PDI ) 1.08) by GPC (Table 1). The 1H NMR spectrum in chloroform-d (CDCl3) showed three peaks derived from the terminal 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). Numberaveraged molecular weight (Mn) and chemical composition of P(IPAAm-co-DMAAm) were determined by 1H NMR, estimating from the integrated proton signals derived from IPAAm methine (4.00 ppm), DMAAm methyl (2.90 ppm), and orthophenyl of terminal dithiobenzoate (7.95 ppm; Mn ) 9300,
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Table 1. Characterization of Polymer Compositions and Molecular Weights polymer sample code
monomer unitsa IPAAm/DMAAm/LA
TBT-P(IPAAm-co-DMAAm)-OH TBT-P(IPAAm-co-DMAAm)-b-PLA
54/29/54/29/14
Mnb
PDIc
9300 1.08 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 mM LiCl.
monomer unit: IPAAm/DMAAm ) 54/29). DMAAm composition in the thermoresponsive polymers was approximately 5 mol % higher than initial feed DMAAm composition, and monomer ratio was controlled by the initial monomer composition.33 The polymers in DMF showed a n-π* absorption band (λmax ) 498 nm) corresponding to terminal dithiobenzoate groups, and Mn by end group analysis was 9600, calculated from the molar extinction coefficient of terminal dithiobenzoate (determined as 109.5 L mol-1 cm-1 in DMF). In addition, Mn calculated from GPC was 10000, in good agreement with the results of polymer end group assays by both 1H NMR and UV spectrometric measurements. In the second step of the polymer synthesis, diblock copolymers with biodegradable segments were prepared by ring-opening polymerization of LA initiated by P(IPAAmco-DMAAm) R-hydroxyl groups using tin(II) 2-ethylhexanoate as the catalyst. ω-Dithiobenzoate groups in RAFT polymers thermally decompose at a temperature over 120 °C.42 Therefore, we optimized the polymerization condition (110 °C, 12 h) and successfully obtained diblock polymers with a high ω-functionality (93%, determined by 1H NMR). The resultant diblock copolymers had a low PDI of 1.07 by GPC. According to 1H NMR studies of the diblock copolymers, Mn and monomer compositions were then determined by the peak areas of IPAAm methine (4.00 ppm), DMAAm methyl (2.90 ppm), and LA methylene (5.20 ppm) protons. Additionally, Mn obtained from GPC was 12000, comparable to the molecular weight determined by 1H NMR. The characterization of these polymers is summarized in Table 1. Aminolysis and Conversion of Polymer Termini. An important feature specific to RAFT polymerization is the possibility to prepare polymers possessing terminal dithioester groups that can be easily aminolyzed with primary or secondary amines, producing terminal thiol groups43,44 (Figure 1b). This feature of RAFT technique has potential for molecular design and preparation of functional polymers including bioconjugated polymers and fluorescently labeled polymers by thiol coupling chemistry.33,38,45 In this work, we attempted substitutions of the corona-forming thermoresponsive polymer termini by a facile maleimide-thiol coupling reaction. TBT-P(IPAAm-coDMAAm)-b-PLA diblock polymers were pink in color due to a n-π* absorption band around 500 nm corresponding to the ω-dithiobenzoate groups. After polymer terminal aminolysis, the absorption completely disappeared. We adopted a one-pot reaction of both ω-dithiobenzoate aminolysis and coupling reaction with maleimide derivatives for highly efficient terminal substitution. GPC elution curves and PDI values of diblock copolymers were comparable before and after the terminal coupling reaction, polymers reacted with maleimide, and its OGderivative showed narrow PDIs of 1.06 and 1.11, respectively. Formation and Characterization of Polymeric Micelles. Core-corona type thermoresponsive polymeric micelles were obtained by dialysis of P(IPAAm-co-DMAAm)/PLA block copolymers in DMAc against water at 10 °C.46 We have previously reported that surface functional groups influenced
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Table 2. Characterization of OG-Labeled Thermoresponsive Polymers and Polymeric Micelles 37 °C b
sample code
LCST (°C)
P(IPAAm-co-DMAAm) M(Mal) M(OG)a
40.2 40.0 39.4
diameter (nm) 8.1 ( 1.6 23.3 ( 12.8 20.7 ( 8.5
42 °C PDI 0.14 NDc 0.20
diameter (nm) 8.4 ( 1.8 613.9 ( 273.6 588.8 ( 73.3
PDI 0.22 NDc 0.041
a 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 polymeric micelles at temperatures below (37 °C, open circle) and above (42 °C, closed circle) the LCST in DPBS(-).
on the micellar LCST.33 In addition, we consider that highly concentrated fluorescent moieties on the micellar surfaces may lead to fluorescent quenching. Therefore, in this work, OGlabeled polymeric micelles were prepared by mixing of Maland OG-P(IPAAm-co-DMAAm)-b-PLA (Mal/OG ) 80/20 in polymer wt %). Obtained P(IPAAm-co-DMAAm)/PLA micelle solutions were highly transparent and shown to have nanoscale diameters and monomodal size distribution at 37 °C, regardless of surface functional group differences (Table 2). In addition, the polymeric micelles showed a monomodal size distribution in the presence of bovine serum albumin (see Figure S5 in Supporting Information), suggesting limited interaction with serum proteins by densely packed thermoresponsive polymer chains of the micelle outer shells. For passive drug targeting using carrier systems, nano-order carrier sizes (ca. 5-200 nm) are strongly desirable for longterm circulation in the bloodstream, avoiding both renal filtration and RES uptake,28 and for subsequent selective tumor accumulation based on specific macroscopic properties of solid tumors, the EPR effect.29,30 We investigated the micellar thermoresponsive behavior in DPBS(-) by measuring optical transmittance changes at various temperatures with results shown in Table 2. P(IPAAm-co-DMAAm)/PLA micelles exhibited a phase transition throughout the LCSTs (ca. 40 °C) based on hydrophilic/hydrophobic switching of the corona-forming thermoresponsive polymer segments. The obtained thermoresponsive polymeric micelles can be utilized as the intelligent drug carrier 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 hydrodynamic diameters increased and formed unimodal submicron-ordered aggregates of 614 and 589 nm, respectively (Table 2 and Figure 2). The polymer aggregation was caused by the alternation of the micellar corona property from hydrophilic to hydrophobic, and subsequent promotion of hydrophobic interaction between thermoresponsive chains in aqueous media. The introduction of OG moieties to the micellar surfaces scarcely affected the
micellar properties including size distribution and thermoresponsive behavior. Cellular Uptake and Intracellular Distribution of the Polymeric Micelles. Effects of differences in temperature throughout the LCST on intracellular uptake of polymer micelles were 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 not show any cytotoxicity and internalize to the cultured cells (data not shown), and considered that influence of OG introduction to the micellar surfaces on cytotoxicity and cellular uptake was negligible. Before the microscopy experiments, the cells incubated with the micelles were gently and thoroughly rinsed with DPBS(-) to remove any micelles adhered to the cell surfaces at 25 °C. In the CLSM images, the green fluorescence derived from the OG-labeled micelles was negligible below the micelle LCST (37 °C, Figure 3a). Of great interest, the fluorescence from micelles inside the cells was clearly observed above the LCST (42 °C), and green-colored dots were localized around the cell nuclei (Figure 3b). We have obtained similar CLSM images of the micelle-treated cells without cytological fixation, indicating that fixation treatments did not affect on the permeability of cellular membranes. We further investigated time dependence and efficiency of temperature-modulated intracellular uptake by flow cytometry. According to the flow cytometric studies, cellular uptake of the micelles incubated at 37 °C (below the LCST) was considerably low after 24 h (Figure 4). By contrast, the micelles incubated at 42 °C (above the LCST) were clearly detected within the cells at 2 h and later. The intensity of OG fluorescence derived from these micelles kept increasing up to 6 h, to approximately 16-fold greater intracellular uptake (Figure 4a). Additionally, we investigated effects of temperature changes on cellular membrane permeability using propidium iodide. Cellular membrane permeability was independent of incubation temperatures ranging from 37 to 42 °C, indicating that cellular uptake of the micelles did not result from changes of membrane permeability at elevated temperature. We further investigated influence of FBS on intercellular uptake of the polymeric micelles. Relative cellular uptake of the micelles without FBS was 74.5 ( 1.1% (n ) 4) compared to that with FBS. In the DLS studies, the micelles did not significantly interact with the serum albumins at temperatures both below and above the LCST (see Figure S5 in Supporting Information). Therefore, the decrease in cellular uptake of the micelles was probably caused by lowered cell growth and viability in absence of FBS. We further investigated effects of the polymer assembly on thermally modulated intracellular localization using OGlabeled P(IPAAm-co-DMAAm) (Mn ) 10300, Mw/Mn ) 1.07, 200 µg/mL). As shown in Figure 4b, cellular uptake of the OGlabeled P(IPAAm-co-DMAAm) was comparable to that of the thermoresponsive micelles at 37 °C (below LCST). In addition, cellular uptake of the thermoresponsive linear polymers was not affected by temperature change throughout the LCST from 37 to 42 °C. No temperature effect on cellular uptake of linear
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Figure 3. CLSM images of polymeric micelles localized within cultured cells after incubation for 9 h (a) below the LCST (37 °C) and (b) above the 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 5. Viabilities of cultured endothelial cells incubated with Mallabeled polymer micelles at various concentrations at 37 °C (open circle) and above 42 °C (closed circle; mean ( S.D., n ) 8). Incubation time: 24 h.
Figure 4. Time-dependent cellular uptake of (a) OG-labeled thermoresponsive micelles and (b) the OG-labeled P(IPAAm-co-DMAAm) at temperatures below (37 °C, open circle) and above (42 °C, closed circle) their LCST (mean ( SD, n ) 4). Y-axis: mean fluorescent intensity of 10000 events.
polymers was observed because dehydrated polymer chains did not aggregate at the polymer concentration of 0.2 mg/mL above the LCST as shown in Table 2. The enhancement of intracellular uptake for the polymeric micelle system seems to be due to two possible mechanisms. First, hydrophobic interactions between the cell membranes and hydrophobic micelle cores were promoted by collapse of the thermoresponsive corona-forming polymers above the LCST. Below the LCST, densely packed and hydrated thermoresponsive corona-forming P(IPAAm-coDMAAm) reduced possible interactions of the PLA cores with cell surfaces. Upon temperature increase above the LCST, the corona-forming polymer chains collapse with the dehydration of IPAAm units. Because of significant polymer conformational changes, hydrophobic interactions between the micelles and cell membranes increase. Consequently, adhesion of the micelles
to the cell surfaces was promoted, followed by enhancement of intracellular uptake. A second possibility is that the corona PIPAAm derivatives attached to the hydrophobic polymeric cores regulate micelle adhesion to cell surfaces and sustained intracellular uptake. We have previously reported that ultrathin PIPAAm-grafted layers (
