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Article Cite This: ACS Omega 2019, 4, 6443−6451
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LAT1-Targeting Thermoresponsive Liposomes for Effective Cellular Uptake by Cancer Cells Minami Maekawa-Matsuura, Kei Fujieda, Yutaro Maekawa, Tomohiro Nishimura, Kenichi Nagase,* and Hideko Kanazawa* Faculty of Pharmacy, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan
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ABSTRACT: L-type amino acid transporter 1 (LAT1) is a transporter that is more highly expressed in cancer cells compared with normal cells. In the present study, liposomes, composed of egg phosphatidylcholine (EPC) and dioleoyl phosphatidylethanolamine, were modified with LAT1-targeting thermoresponsive polymer, L-tyrosine-conjugated poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-coDMAAm)). The cellular uptake of the prepared LAT1-targeting liposomes was evaluated using HeLa cells as a cancer cell model. At temperatures above the polymer’s lower critical solution temperature, uptake of the liposomes into cells was observed because the polymer at the liposome surface became hydrophobic and interacted with the cell membrane. Flow cytometry analysis suggested that L-tyrosine-P(NIPAAm-co-DMAAm)-liposomes exhibited markedly increased cellular uptake by HeLa cells compared with that of liposomes not modified with L-tyrosine. This result indicated that cellular uptake of liposomes can be enhanced by the affinity between L-tyrosine and the LAT1 of HeLa cells. The developed functional liposomes, which exhibit both thermoresponsive and LAT1-targeting properties, would be appropriate for temperature-modulated drug delivery and imaging with good targeting ability.
1. INTRODUCTION Theranosticsthe fusion of diagnosis and therapyhas recently been attracting attention in cancer therapy.1 Cancer cell metabolism is different to that of normal cells,2 and various targets specific to cancer cells are being studied for diagnosis and therapy. Similarly to glucose transporter 1 (GLUT1),3 Ltype amino acid transporter 1 (LAT1),4 L-type amino acid transporter 3,5 and system ASC transporter 26 show increased expression in cancer cells. LAT1 recognizes the charges of the α-amino group and α-carboxyl group of amino acids7 and selectively transports branched and/or aromatic amino acids, for example, tyrosine, leucine, and phenylalanine. LAT1 is highly expressed in tumor tissues in the brain, lung, colon, breast, glia, prostate, and pancreas,8−11 compared with normal cells.12,13 Overexpression is also a prognostic factor for metastasis. 14 Positron emission tomography (PET) probes15−17 have been developed as a diagnostic method for targeting LAT1. In particular, L-[3-18F]-α-methyltyrosine (18FFAMT) has a lower background in normal tissues than 2deoxy-2-[18F] fluoro-D-glucose (18F-FDG) targeting GLUT1,18 suggesting the possibility of PET diagnosis with high tumor selectivity.19 LAT1 is also involved in the accumulation at the tumor site of L-3-[123I]-α-methyl tyrosine used in single photon emission computed tomography19 and L-p-boronophenylalanine used in boron neutron supplement therapy.20 In addition, it has been reported that antitumor effects can be obtained by inhibiting LAT1,21 and LAT1-targeting drug carriers22 have also been studied. However, since transporters expressed in © 2019 American Chemical Society
some normal cells of the blood brain barrier and placenta also recognize these probes and drug carriers, it is important that they exhibit active targeting to act selectively in the target cells after accumulation in the target tumor tissue. Responses to physical stimuli such as temperature,23,24 light,25,26 magnetism,27 and ultrasound,28,29 and chemical stimuli such as pH30,31 and glucose concentration, have been studied for utilization in active targeting. The first report of liposomes with temperature-controlled properties described the gel−liquid crystal phase transition of the liposome membrane.23 Subsequently, a functional liposome32 modified with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), has been studied. PNIPAAm is the most utilized thermoresponsive polymer in biomedical fields.33−44 PNIPAAm exhibits a phase transition attributed to the hydration and dehydration across its lower critical solution temperature (LCST) of 32 °C, leading to hydrophobicity changes. Additionally, the LCST can be modulated by incorporation of co-monomers in the polymerization.45 In drug delivery systems and imaging agent, interaction drug carriers or fluorescent probes with cell membrane can be controlled by changing the polymer properties as a result of temperature.46−50 In particular, thermoresponsive liposomes prepared by modifying the liposomes with thermoresponsive Received: January 23, 2019 Accepted: March 25, 2019 Published: April 8, 2019 6443
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Figure 1. LAT1-targeting thermoresponsive liposomes.
Scheme 1. Synthesis of DOPE-Modified Thermoresponsive Polymers
raphy) of the P(NIPAAm-co-DMAAm) was 16 700 and that of the Tyr-P(NIPAAm-co-DMAAm) was 12 100.48 The conjugation of the tyrosine was confirmed by 1H NMR.48 The terminal ends of these temperature-responsive polymers were conjugated with DOPE (Scheme 1). The modification of DOPE to these polymers was confirmed by 1H NMR (Figure S1). To investigate the effect of the conjugation of L-tyrosine and DOPE to the thermoresponsive polymers, on their thermoresponsive properties, the phase transition behavior of the polymers was observed by differential scanning calorimetry (DSC) (Figure 2). An endothermic peak associated with phase transition was observed for all of the prepared polymers. The endothermic peak values for P(NIPAAm-co-DMAAm) and Tyr-P(NIPAAm-co-DMAAm) were at 37.9 and 37.1 °C, respectively (Figure 2a). These results indicated that the conjugation of L-tyrosine to thermoresponsive polymer did not affect the thermoresponsive properties. Additionally, the endothermic peak value of the polymers after DOPE conjugation was determined (Figure 2b). The values for P(NIPAAm-co-DMAAm)-DOPE and Tyr-P(NIPAAm-coDMAAm)-DOPE were at 37.6 and 38.0 °C, respectively. The difference in endothermic peak temperatures before and after modification of DOPE was within 1 °C, indicating that DOPE conjugation to the thermoresponsive polymers does not affect the thermoresponsive properties. Temperature-responsive liposomes composed of DOPEmodified thermoresponsive polymer (P(NIPAAm-co-
polymers are promising drug and gene carriers because of their excellent stability and their drug and gene carrying capacity.51−56 In addition, because liposomes can be passively accumulated in tumor tissues as a result of the enhanced permeability and retention effect,57 a synergistic effect is expected as a result of optimizing particle size.58 We have already reported that the cellular uptake of LAT1targeting thermoresponsive fluorescent polymer probes by HeLa cells was enhanced compared with that of probes without LAT1-targeting amino acids.48 Therefore, if LAT1targeting amino acids are used to modify thermoresponsive liposomes, further functional thermoresponsive liposomes with cancer cell targeting properties could be developed. In this study, liposomes composed of 3-sn-phosphatidylcholine, from egg yolk, (EPC) and dioleoyl phosphatidylethanolamine (DOPE) were modified with thermoresponsive polymer with a LAT1 affinity site. The temperature-dependent and LAT1 affinity site-selective cellular uptake properties of the prepared liposomes were investigated (Figure 1).
2. RESULTS AND DISCUSSION 2.1. Preparation of Functional Liposomes. The thermoresponsive polymer, P(NIPAAm-co-DMAAm), containing 20 mol % DMAAm, and the thermoresponsive polymer with LAT1-targeting amino acid, L-tyrosine-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) [Tyr-P(NIPAAm-coDMAAm)], were prepared as previously reported.48 The molecular weight (determined by gel permeation chromatog6444
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DMAAm)-DOPE or Tyr-P(NIPAAm-co-DMAAm)-DOPE), EPC, and DOPE were prepared. Unmodified liposomes were prepared as a comparative control group. Additionally, polyethylene glycol (PEG)-modified liposomes were also prepared and used as a control because PEG-modified liposomes have been widely used as drug carriers in previous reports.59 The lipid composition of the liposomes was EPC/ DOPE (1:1, molar ratio). The liposomes were characterized by measuring phase transition profiles (Figure 3a), particle size (Table 1 and Figure 3b,c), and zeta potential (Table 1). The unmodified liposomes and PEG-modified liposomes showed no change in transmittance. In contrast, the thermoresponsive polymer-modified liposomes showed a dramatic decrease in transmittance around 40 °C (Figure 3a). This is because the thermoresponsive polymer-modified liposomes dehydrated and became hydrophobic as a result of a phase transition of the polymer around 38 °C. The polymer-modified liposomes aggregated with each other through hydrophobic interactions and precipitated, leading to a decrease in the transmittance of the liposome suspension. The results indicated that the temperature-responsive liposomes change their hydrophobicity near body temperature. The particle size of the prepared liposomes was in the range 130.5−145.1 nm at 25 °C (Table 1), indicating that the prepared liposomes were a suitable size for cellular uptake and circulation in vivo as previously reported.58 The PDI of the prepared liposomes was 0.12 or less, indicating that the prepared liposomes have a uniform size. The size of the thermoresponsive polymer-modified liposomes increased when they were heated above the LCST (Figure 3b), while the PEG-modified and unmodified liposomes maintained their size. This is because the
Figure 2. Microcalorimetric endotherms of (a) P(NIPAAm-coDMAAm) and Tyr-P(NIPAAm-co-DMAAm) and (b) P(NIPAAmco-DMAAm)-DOPE and Tyr-P(NIPAAm-co-DMAAm)-DOPE.
Figure 3. (a) Temperature dependence of liposome transmittance, (b) effect of temperature on liposome particle size, (c) reversible particle size changes for thermoresponsive polymer modified-liposomes with successive heating (45 °C; closed markers) and cooling (37 °C; open markers) cycles. 6445
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Table 1. Properties of the Prepared Liposomes size (nm)a unmodified liposome PEG-modified liposome P(NIPAAm-co-DMAAm)-liposome Tyr-P(NIPAAm-co-DMAAm)-liposome
145.1 144.0 141.8 130.5
± ± ± ±
3.1 2.5 1.1 0.5
PDIa 0.05 0.04 0.12 0.06
zeta potential (mV)a −11.9 −5.6 −6.4 −8.1
± ± ± ±
0.9 1.6 0.8 0.4
Determined by DLS. Mean ± SD, n = 3.
a
thermoresponsive polymer on the liposome becomes hydrophobic above the LCST, and the polymer-modified liposomes aggregate with each other through hydrophobic interaction. Therefore, the apparent liposome size increased with increasing temperature above the LCST. To confirm the repeatability of the temperature response of the liposomes, the liposome size was observed while repeatedly cycling the temperature (Figure 3c). When the temperature was varied between 45 and 37 °C, which are above and below the LCST, respectively, in successive cycles, the particle size showed a reversible change, that increasing above the LCST and decreasing below the LCST. These results showed that the prepared liposomes show reproducible temperature responses. The zeta potential of the liposomes was measured in order to investigate the electrostatic properties. The observed zeta potentials were negative for all of the liposomes, which is attributed to the anionic nature of the phosphate group of the lipids. Modifying the liposomes with PEG or thermoresponsive polymer reduced the anionic properties of the liposomes because the presence of the polymer had a shielding effect. 2.2. Microscopy Observation of Liposome Uptake into Cells. To investigate the effect of temperature and LAT1 recognition on liposome uptake, 5(6)-carboxyfluorescein (CF)-containing liposomes were incubated with HeLa cells and uptake behavior was observed. Unmodified liposomes, PEG-modified liposomes, P(NIPAAm-co-DMAAm)-liposomes, or Tyr-P(NIPAAm-co-DMAAm)-liposomes were added to HeLa cells that were incubated for 4 h at 37 or 42 °C. After incubation, confocal laser scanning microscopy observation was carried out (Figure 4). Confocal microscopy images indicated that slight fluorescence, indicating cellular uptake, was observed for PEG-modified liposomes and P(NIPAAm-co-DMAAm)-liposomes at 37 °C (Figure 4b,c). In contrast, cellular uptake of all liposomes was confirmed at 42 °C. In particular, greater fluorescence was observed for liposomes modified with Tyr-P(NIPAAm-co-DMAAm) than for the other liposomes (Figure 4d). To investigate the localization of the liposomes in the cells, lysosomes were also stained and liposome localization was observed by confocal laser scanning microscopy at 37 and 42 °C (Figure 5). At 42 °C, liposomes were observed near the lysosome, while liposomes were not observed near the lysosome at 37 °C. These results suggest that the cellular uptake of liposomes was enhanced by external temperature change. The number of PEG-modified and P(NIPAAm-coDMAAm)-liposomes in lysosomes increased at 42 °C compared with that of unmodified liposomes. However, fluorescence not localized in lysosomes was also observed for PEG-modified liposomes and P(NIPAAm-co-DMAAm)-liposomes (Figure 5b,c). In comparison, above the LCST, almost all of the Tyr-P(NIPAAm-co-DMAAm)-liposomes were localized in the lysosome (Figure 5d). This result suggested that Tyr-P(NIPAAm-co-DMAAm)-liposomes can be more
Figure 4. Microscopy images of HeLa cells after a 4 h incubation with (a) unmodified liposomes, (b) PEG-modified liposomes, (c) P(NIPAAm-co-DMAAm)-liposomes, and (d) Tyr-P(NIPAAm-coDMAAm)-liposomes at 37 and 42 °C. Images were obtained by confocal laser scanning microscopy. Nuclei are stained blue (DAPI) and CF appears green. Scale bar represents 20 μm.
effectively taken up by cells compared with liposomes modified with P(NIPAAm-co-DMAAm). 2.3. Flow Cytometry Analysis of Liposomes Uptake into Cells. To evaluate the uptake efficiency of the liposomes into cells, the fluorescence intensity of the HeLa cells was analyzed by flow cytometry. CF-containing liposomes were incubated with HeLa cells at 37 and 42 °C and fluorescent intensity was measured (Figure 6). Cells treated with unmodified liposomes and PEG-modified liposomes exhibited little difference in fluorescence intensity compared with the control (Figure 6a,b). This result indicates that most of these liposomes were not taken up into cells, leading to low fluorescence intensity. Increased fluorescence intensity was observed for cells treated with liposomes modified with P(NIPAAm-co-DMAAm) compared with those treated with unmodified and PEG-modified liposomes (Figure 6c), which indicates that P(NIPAAm-co-DMAAm)-liposomes were taken up by cells. In addition, the fluorescence intensity of the P(NIPAAm-co-DMAAm)-liposome treated cells increased at 42 °C compared with those incubated at 37 °C because the 6446
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DMAAm)-liposomes exhibited the largest increase in fluorescence intensity compared with the control (Figure 6d). The high fluorescence intensity indicated that Tyr-P(NIPAAm-coDMAAm)-liposomes were more effectively taken up into cells than all other liposomes. In addition, fluorescence intensity between cells incubated at 37 and 42 °C was quite different for the Tyr-P(NIPAAm-co-DMAAm)-liposome case compared with P(NIPAAm-co-DMAAm)-liposome treated cells. This result indicates that the terminal L-tyrosine of Tyr-P(NIPAAmco-DMAAm) induced enhanced uptake above the LCST. To investigate the time-dependent uptake of the liposomes, the fluorescence intensity of the cells was observed over a time course (Figure 7). Cells incubated with unmodified liposomes and PEG-modified liposomes exhibited a slight difference in fluorescence intensity between 37 and 42 °C over a time course of 4 h (Figure 7a,b) because the liposomes did not have thermoresponsive properties. The fluorescence intensity of the cells treated with P(NIPAAm-co-DMAAm)-liposomes showed a difference between 37 and 42 °C, and the difference increased with increasing incubation time (Figure 7c). This result indicates that the cellular uptake of P(NIPAAm-coDMAAm)-liposomes was enhanced by increasing the incubation time. In contrast, the fluorescence intensity of cells treated with Tyr-P(NIPAAm-co-DMAAm)-liposomes markedly increased with incubation time at 42 °C, while the fluorescence intensity at 37 °C remained constant over the incubation period (Figure 7d). In addition, a greater difference between the fluorescence intensity at 37 and 42 °C was observed for cells treated with Tyr-P(NIPAAm-co-DMAAm)liposomeseven after the initial incubation periodcompared with the difference for P(NIPAAm-co-DMAAm)-liposome treated cells. This result suggests that the synergetic effect of the hydrophobic interaction between the polymer and the cell membrane, and the affinity between the polymer terminal L-tyrosine and LAT1 of cells, enhances the cellular uptake of the liposomes.
Figure 5. Microscopy images of HeLa cells after a 4 h incubation with (a) unmodified liposomes, (b) PEG-modified liposomes, (c) P(NIPAAm-co-DMAAm)-liposomes, and (d) Tyr-P(NIPAAm-coDMAAm)-liposomes at 37 and 42 °C. Nuclei are stained blue (DAPI), CF appears green, and lysosomes are stained red (LysoTracker Red DND-99). Scale bar represents 20 μm.
P(NIPAAm-co-DMAAm) of the liposomes became hydrophobic, leading to enhanced uptake at 42 °C.55,56 Of all liposome-treated cells, those treated with Tyr-P(NIPAAm-co-
Figure 6. Histograms of flow cytometry. The fluorescence intensity of HeLa cells after a 4 h treatment with (a) unmodified liposomes, (b) PEGmodified liposomes, (c) P(NIPAAm-co-DMAAm)-liposomes, and (d) Tyr-P(NIPAAm-co-DMAAm)-liposomes at 37 °C (blue) and 42 °C (red). Gray shows a control of HeLa cells without incubation with liposomes. 6447
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Figure 7. Fluorescence intensity of HeLa cells over time (0.5, 1, 2, and 4 h) following incubation with (a) unmodified liposomes, (b) PEGmodified liposomes, (c) P(NIPAAm-co-DMAAm)-liposomes, and (d) Tyr-P(NIPAAm-co-DMAAm)-liposomes at either 42 °C (closed circles) or 37 °C (open circles). The fluorescence intensity of each liposome incubated at 37 °C for 0.5 h was defined as 1.0. Data are mean ± standard deviation (n = 3) *p < 0.05.
LCST and was not observed below the LCST. Liposome uptake into cells was found to proceed by endocytosis, as the liposomes incorporated into cells were localized in lysosomes. Flow cytometry analysis indicated that the fluorescence intensity of cells incubated with L-tyrosine-conjugated P(NIPAAm-co-DMAAm)-liposomes was larger than that of cells incubated with liposomes not modified with L-tyrosine. The prepared LAT1-targeting thermoresponsive liposomes would be useful drug carriers for encapsulating anticancer drugs such as doxorubicin because they have selectivity for cancer cells owing to their LAT1 affinity. In addition, the uptake of the liposomes by cells could be regulated through changes in their hydrophobicity resulting from variations in temperature. Thus, the developed liposomes could be used as functional drug carriers with LAT1 and temperature responsiveness.
Additionally, there was a slight difference in the uptake fluorescence detected for the microscope observation (Figures 4 and 5) compared with the flow cytometry analysis (Figure 7). This is attributed to the nonspecific adsorption of liposomes on the cell membrane and cell culture dish. In the fluorescence microscope observation, liposomes adsorbed on the cell membrane and cell culture dish were observed although the cells were rinsed gently using ethylenediaminetetraacetate (EDTA) solution. In contrast, in the flow cytometry analysis, cells were recovered from the cell culture dish using trypsin and recovered cells were rinsed with minimum Eagle’s essential medium (MEM) containing 10% fetal bovine serum (FBS), 1 mM EDTA in phosphate-buffered saline (PBS), and PBS. Therefore, liposomes adsorbed on the cell membrane were removed, and only liposomes taken up by cells were detected by flow cytometry analysis. The synthesized thermoresponsive polymer-modified liposomes exhibited temperature and LAT1 affinity-dependent cellular uptake. Therefore, the LAT1-targeting temperatureresponsive polymer-modified liposomes would be useful for temperature-modulated imaging and drug delivery, with the ability to target malignant tumors involved in LAT1 expression and respond to the local environment of cancer cells.
4. MATERIALS AND METHODS 4.1. Materials. N-(3-Maleimide-1-oxopropyl)-dioleoyl phosphatidylethanolamine (DOPE-maleimide) was purchased from NOF (Tokyo, Japan). EPC and DOPE were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). 1,2Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DHPE-PEG) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). CF was obtained from Sigma-Aldrich (St. Louis, MO, USA). 4.2. Synthesis of DOPE-Modified Thermoresponsive Polymers. P(NIPAAm-co-DMAAm) and Tyr-P(NIPAAm-coDMAAm) containing 20 mol % DMAAm were synthesized as previously reported.48 P(NIPAAm-co-DMAAm) (100 mg, 6.7 × 10−3 mmol) was dissolved in tetrahydrofuran (THF; 4 mL).
3. CONCLUSIONS In the present study, CF-containing liposomes, which were recognized by LAT1 and were taken up by cells in response to temperature, with high affinity for cancer cells, were developed and evaluated by microscopy and flow cytometry analysis. Cellular uptake of the liposomes was confirmed above the 6448
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Then, 2 mL of 2-aminoethanol solution in THF (4.0 μL, 6.7 × 10−2 mmol) was added slowly and purged with nitrogen gas. DOPE-maleimide solution in THF (12.2 mg, 6.7 × 10−3 mmol, 2 mL) was added and the mixture was reacted. The reaction was proceeded under an atmosphere of nitrogen for one day. The solution was purified by dialysis using a dialysis membrane (MWCO: 3500). The product was dried to provide P(NIPAAm-co-DMAAm)-DOPE (94 mg) as a white solid. Tyr-P(NIPAAm-co-DMAAm)-DOPE (96 mg) was synthesized from Tyr-P(NIPAAm-co-DMAAm) (100 mg) using the same procedure. 4.3. Preparation of the Liposomes. EPC (7.5 mg, 47.5 mol %), DOPE (7.5 mg, 47.5 mol %), and P(NIPAAm-coDMAAm)-DOPE (19.6 mg, 5 mol %) were dissolved in CHCl3 (2.5 mL) and the solvent was evaporated to obtain a lipid film. After replacing with nitrogen, 3.0 mM CF in PBS solution (1 mL) was added and ultrasonic disruption was carried out for 30 min to peel off the lipid film. The solution was homogenized through polycarbonate membrane filters with 0.2 μm pore size and purified through a PD-10 column (GE Healthcare Bio-Sciences AB, Sweden), and P(NIPAAmco-DMAAm)-liposomes (lipid concentration: 6.48 mg/mL) were then obtained as a white liquid. Tyr-P(NIPAAm-coDMAAm)-liposomes (lipid concentration: 6.23 mg/mL) were obtained from EPC (7.5 mg, 47.5 mol %), DOPE (7.5 mg, 47.5 mol %), and Tyr-P (NIPAAm-co-DMAAm)-DOPE (14.4 mg, 5 mol %) using the same procedure. Unmodified liposomes (lipid concentration: 7.02 mg/mL) were obtained from EPC (7.5 mg, 50 mol %) and DOPE (7.5 mg, 50 mol %). PEG-modified liposomes (lipid concentration: 7.42 mg/mL) were obtained from EPC (7.5 mg, 47.5 mol %), DOPE (7.5 mg, 47.5 mol %), and DHPE-PEG (3.2 mg, 5 mol %). Lipid concentration was determined using Laboratory Assay Phospholipid (FUJIFILM Wako Pure Chemical). 4.4. DSC Measurement. The DSC of DOPE-modified polymers was measured to determine the change in calorific value due to temperature change in PBS solution (30 w/v %) using a differential scanning calorimeter (DSC-60 Plus, Shimadzu, Kyoto, Japan). The atmosphere was controlled with FC-60A (Shimadzu), with the heating rate of 2.5 °C/min. 4.5. Phase Transition Behavior. The phase transition behavior of the liposomes was measured from the optical transmittance at 500 nm in PBS solution (lipid concentration: 0.5 mg/mL) over a range of temperatures at a rate of 0.1 °C/ min using an ultraviolet−visible spectrophotometer (V-630, JASCO, Tokyo, Japan). 4.6. Particle Size Measurement. The particle size measurement of the prepared liposomes was performed by DLS using a Zetasizer Nano ZS (Malvern Panalytical, Grovewood Road, Malvern, UK). The liposomes were dispersed in PBS (lipid concentration: 0.1 mg/mL) and the temperature dependence of liposome size was evaluated between 25 and 50 °C. 4.7. Zeta Potential. The zeta potential of the liposomes was measured by electrophoretic light scatting using a Zetasizer Nano ZS. The liposome suspensions were dispersed in PBS (lipid concentration: 0.5 mg/mL) and measured at 25 °C. 4.8. Cell Culture. HeLa cells were obtained from RIKEN BRC (Ibaraki, Japan) and cultured using MEM as a base medium, containing 10% fetal bovine serum, 50 μg/mL streptomycin, 50 units/mL penicillin, and 146 μg/mL Lglutamine at 37 °C in 5% CO2.
4.9. Confocal Microscopy Observation. A suspension of HeLa cells (1.0 × 105 cells/0.5 mL) was added to each well of a 4-well culture slide (Thermo Fisher Scientific) and incubated overnight. CF-containing liposomes (50 μg) were then added to the cells and further incubation was performed for 4 h at 37 or 42 °C. Cells were rinsed with 1 mM EDTA in PBS. LysoTracker Red DND-99 (Thermo Fisher Scientific) in MEM (50 μM) was added to the cells, and subsequent incubation was performed at 37 °C for 30 min. The cells were rinsed using EDTA solution twice and then fixed with 4% paraformaldehyde in PBS for 20 min. Cells were rinsed with PBS, and coverslips were put on the cells in a mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). The prepared samples were observed with a confocal laser microscope (FV1000D, Olympus, Tokyo, Japan). 4.10. Flow Cytometry Analysis. A suspension of HeLa cells (2.0 × 105 cells/2 mL) was added to each well of a 6-well plate. Cells were incubated with CF-containing liposomes (100 μg) at 37 or 42 °C for 0.5, 1, 2, or 4 h. The cells were then rinsed with 1 mM EDTA in PBS. The cells were recovered with trypsin and rinsed with MEM containing 10% FBS, 1 mM EDTA in PBS, and PBS. The cell suspension was filtered using a 35 μm nylon mesh. Flow cytometry analysis was performed using LSRII (BD, Franklin Lakes, NJ, USA). Fluorescence histograms were obtained using FACSDiva software. Statistical analysis of obtained data was performed using Student’s t-test. The fluorescence intensity of each liposome incubated at 37 °C for 0.5 h was defined as 1.0.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00216. 1 H NMR spectrum of DOPE-modified thermoresponsive polymers (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (K.N.). *E-mail:
[email protected] (H.K.). ORCID
Kenichi Nagase: 0000-0002-6575-0107 Hideko Kanazawa: 0000-0003-2550-470X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The study was partially supported by the Strategic Research Foundation at Private Universities, S1411004. REFERENCES
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