Colloids and Surfaces B: Biointerfaces 128 (2015) 94–99
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Trading polymeric microspheres: Exchanging DNA molecules via microsphere interaction Nobuyuki Morimoto a,∗ , Kanna Muramatsu a , Shin-ichiro M. Nomura b , Makoto Suzuki a a
Department of Materials Processing, Graduate School of Engineering, Tohoku University, 6-6-02 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, 6-6-01 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan b
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 18 December 2014 Accepted 8 February 2015 Available online 17 February 2015 Keywords: Zwitterionic block copolymers Self-assembly Microspheres Single-stranded DNA oligomer Double strand formation
a b s t r a c t A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole–dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1 m) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3 -TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5 -BHQ-2labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the fundamental aspects of signal transduction and molecule delivery in cell–cell communication, contact-dependent signaling plays important roles, especially in immune and developmental systems and in the control of cell growth, differentiation, and morphogenesis [1]. The paracrine system and signaling via gap junctions are the major pathways of contact-dependent signaling. In paracrine signaling, the paracrine factors of signal molecules released from a cell diffuse locally to neighboring cells. Specific receptors exchange the signals to control the cell’s internal conditions. In contrast, signaling via gap junctions between contacted cytosols is facilitated diffusion. The gap junction intercellular channel regulates the transport of molecules by their molecular weight. In both types of signaling pathways, diffusion of signaling molecules is skillfully regulated. Biomimicking such contact-dependent transport system has led to the design of novel artificial microreactors, sensor devices and drug delivery systems. Polymeric microspheres, comparable in size to living cells, have been designed not only solid spheres but also hollow spheres
∗ Corresponding author. Tel.: +81 22 795 7365; fax: +81 22 795 7313. E-mail address: [email protected] (N. Morimoto). http://dx.doi.org/10.1016/j.colsurfb.2015.02.014 0927-7765/© 2015 Elsevier B.V. All rights reserved.
or vesicles [2–4], hydrogels [5,6], coacervates [7–10], and so on. There are many reports of controlling the release of encapsulated molecules in microspheres by simple diffusion or external stimuli [6,11–14]. However, the efficiency of the transport of encapsulated molecules between microspheres is expected to be low because of the simple, uncontrollable diffusion of the released molecules. In addition, the released molecules could be encapsulated into other microspheres under the same conditions with release in the absence of receptors. Therefore, construction of a contactdependent transport system would improve the effective transport of molecules between microspheres. Poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PDMAPS is known for its biocompatibility [15,16] and thermoresponsiveness of upper critical solution temperature (UCST) [17–19]. Those properties are derived from betaine structure of PDMAPS in the side chain. Below the UCST, the PDMAPS forms an unstable aggregate by the dipole–dipole interaction of the sulfobetaine units. Self-assembled nanoparticles have been designed by controlling the dipole–dipole interaction in water [20]. In our previous study, we prepared a block copolymer of poly(ethylene glycol)-b-PDMAPS (PEG-b-PDMAPS) via reversible addition fragmentation transfer (RAFT) polymerization [21]. The PEG-b-PDMAPS formed self-assembled and multi-layered microspheres in cold water. The PEG block improved the colloidal
N. Morimoto et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 94–99
stability of PDMAPS without losing the thermoresponsiveness. PEG-b-PDMAPS microsphere was controllable against dissociation above their UCST, as well as by the addition of salt. We report here the distinctive interaction between PEG-b-PDMAPS microspheres and the transport properties of encapsulated molecules using single-stranded DNA oligomers (ssDNA) as a model. 2. Experimental 2.1. Materials PEG-b-PDMAPS was prepared as reported previously [21]. The molecular weight of PEG-b-PDMAPS polymers were characterized by 1 H NMR and gel permeation chromatography (GPC) as shown in Table S1. 5 -Carboxymethylrhodamine (TAMRA) or 3 -Black Hole Quencher-2 (BHQ-2) modified DNA oligomers were purchased from FASMAC (Kanagawa, Japan). The sequences were as follows: TAMRA – 5 -TGT GGT ATG GCT GAT TAT GA-3 BHQ-2 – 3 -ACA CCA TAC CGA CTA ATA CT-5 The DNA oligomers were purified by high-performance liquid chromatography (HPLC) and analyzed by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectroscopy. Other reagents were purchased from Sigma–Aldrich (St. Louis, MO) and were used without further purification. 3. Methods
95
(IX-71, Olympus, Tokyo, Japan) equipped with a complementary metal–oxide–semiconductor (CMOS) camera (Neo 5.5 sCMOS, Andor, Northern Island, UK). 3.4. Evaluation of double strand formation Double strand formation of complementary ssDNA encapsulated in PEG-b-PDMAPS was evaluated as evidence of interaction between microspheres. Complementary ssDNA, one of modified fluorophore TAMRA in the 3 -terminus and the other of modified BHQ-2 in the 5 -terminus, were separately encapsulated. The ssDNA encapsulated microsphere solutions were mixed at r.t. after which the mixture was incubated at test temperature with a shaking rate of 60 min−1 . Double strand formation was evaluated by a decrease of the fluorescence intensity of TAMRA. The microsphere mixture was added to 150 mM (final concentration) NaCl solution containing 30 times excess (final concentration) of complementary ssDNA without TAMRA immobilization to inhibit unnecessary quenching. Fluorescence spectra were recorded with a fluorescence spectrometer (F-2500, Hitachi, Tokyo, Japan). The excitation wavelength was 547 nm, and spectra were recorded from 550 to 700 nm. The value of relative fluorescent intensity was calculated by the following equation: Relative fluorescent intensity =
I(t) − I(q) I(0) − I(q)
where I(0) is initial fluorescent intensity, I(t) is that at time t, and I(q) is that after quenching by addition of NaCl solution in the absence of excess complementary ssDNA.
3.1. Preparation of PEG-b-PDMAPS microspheres 4. Results and discussion PEG-b-PDMAPS (final concentration of sulfobetaine unit in polymer: 10 mM) was dispersed in pure water and the solution heated to 70 ◦ C. After 10 min, the solution was cooled to 20 ◦ C for 30 min. For encapsulating the DNA oligomers, DNA solution was mixed with polymer solution at r.t. and then the mixture was annealed by the same treatment as with the polymer solution. All DNA experiments except UV measurements were performed at 10 M phosphates in the ssDNA. 3.2. Characterization of microspheres The size and distribution of the PEG-b-PDMAPS microspheres were evaluated with a high performance laser diffraction analyzer (LA-950, HORIBA, Kyoto, Japan). The PEG-b-PDMAPS microsphere solution was dropped into the chamber cells in the proper scattering intensity. The solution was measured at 25 ◦ C. Zeta potential of PEG-b-PDMAPS microspheres was evaluated by an electrophoretic light scattering analyzer (SZ-100, HORIBA) using a disposable cell equipped with a carbon electrode. The temperature dependence of the cloud point in the PEG-b-PDMAPS microsphere solutions was monitored by UV–vis spectroscopy using a spectrometer (V-630, JASCO, Tokyo, Japan) equipped with a Peltier temperature control unit. The PEG-b-PDMAPS microsphere solution (10 mM in sulfobetaine unit) in the presence or absence of ssDNA was monitored at 550 nm under heating from 5 ◦ C to 65 ◦ C at a heating rate of 1 ◦ C min−1 . 3.3. Microscopic observation The microspheres were optically observed by use of a microscope. A drop of microsphere solution was covered by slide glass and lightly sealed. The specimen was observed and recorded with 10 fps at r.t. Fluorescence-labeled ssDNA encapsulated in PEG-bPDMAPS microspheres was observed by fluorescent microscope
4.1. Interaction between microspheres The characterization and solution properties of PEG-b-PDMAPS and the microspheres used in this study are shown in Table S1 and Table 1, respectively. PEG-b-PDMAPS was coded by the polymerization degrees of PEG and PDMAPS such as 110-73. First, narrow size distributed microspheres with a size of ∼1 m were confirmed in the 110 series of PEG. The longer PEG chain in PEG-b-PDMAPS was assumed to prevent aggregation and supply the colloidal stability of microsphere. The longer PEG chain increased the PEG composition of PEG-b-PDMAPS that increased the hydrophilicity of microsphere. Also the longer PEG-chain might work the excluded volume effect at the outer surface of microspheres. Another possibility was enhancing the phase separation effect between the longer PEG chain and PDMAPS chain in different PEG-b-PDMAPS. On the other hand, the size of 44-81 was not determined because the measured temperature was higher than the UCST. Fig. 1 shows time-lapse images of 110-73 microspheres dispersed in water at 25 ◦ C. Following the time course of the microsphere solution with steps of 0.1 s, contact, fusion and separation of the microspheres were observed in many places, as indicated by the arrows in Fig. 1. There were primarily three kinds of behaviors while the microspheres were in contact. (i) Two microspheres were fused, and then the fused microsphere was immediately (∼0.2 s) split into 2 microspheres Table 1 Solution properties of PEG-b-PDMAPS microspheres at 25 ◦ C. Polymer
Size [m]
Zeta-potential [mV]
Cloud point [◦ C]
22-74 44-81 110-61 110-73 110-119
2.30 ± 0.6 N.D. 0.94 ± 0.1 1.16 ± 0.1 0.78 ± 0.2
−27.9 −36.7 −33.1 −33.7 −36.8
48.5 23.6 37.0 42.0 28.9
± ± ± ± ±
3.4 1.6 4.4 1.8 0.6
± ± ± ± ±
1.7 3.3 2.9 1.7 2.9
96
N. Morimoto et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 94–99
Fig. 1. Microscopic observation of interaction between PEG-b-PDMAPS (110-73) microspheres in water at 25 ◦ C. The arrows indicated the contact of microspheres. Time 0: start of observation.
again with a budding-like behavior. In other cases, 2 microspheres were in contact and formed a peanut-like structure. Then, they (ii) rotated as a unit or (iii) rotated around another microsphere. In all cases, no remarkable size change was observed before and after the contact. The zeta potential of the microspheres was measured to analyze the contact behavior between the microspheres. Interestingly, all microspheres exhibited negative zeta potentials although the chemical structure of PEG-b-PDMAPS showed neutral (Table 1). These solutions exhibited neutral above (>10 ◦ C) the UCST or adding NaCl (>100 mM), when the microspheres were completely dissociated. Sulfobetaine side chain in a PDMAPS unit can form three types of cyclic conformations in water. These are: (1) formation between cationic and anionic groups in a monomer residue (intragroup), formation by head-to-tail stacking of the cationic and anionic groups, (2) within a single molecule (intrachain), or (3) between the neighboring macromolecules (interchain). These heterogenous combinations of betaine side chains might enable the microspheres to have a negative charge. Aside from the electrostatic repulsion, dipole–dipole interaction and van der Waals forces acted as attractive forces between microspheres. These attractive and repulsive forces were also intricately related to Brownian motion of microsphere. Moreover, PEG chain increased dispersion stability of microspheres, and also the multilayered structure of the microsphere might contribute to prevent from their fusion. These multiple factors would have caused the interesting interaction between microspheres. 4.2. Exchanging DNA molecules via microsphere interaction The unique behaviors of contact between microspheres were then applied to transport the encapsulated molecules in the microspheres. Here, we applied double strand formation by 2 kinds of microspheres those were separately encapsulated complementary single-stranded DNA oligomers (ssDNA, 20 mer). PDMAPS is known to interact with DNA [22,23]. First, we tried to encapsulate ssDNA into microspheres. PEG-b-PDMAPS microsphere solutions were heated to 70 ◦ C to dissociate microsphere, and then cooled to 20 ◦ C to form microsphere again. Fig. 2(a) shows a phase contrast microscopic image (upper) and fluorescent microscopic image (lower) of TAMRA-labeled ssDNA encapsulated in PEG-bPDMAPS (110-73) microspheres with a sulfobetaine (correspond to cation)/phosphate ([DMAPS]/[P]) ratio of 1000. No aggregation was confirmed by the encapsulation of ssDNA into microspheres. Though Brownian motion made difficult to observe, most of the microspheres were confirmed as having encapsulated TAMRAlabeled ssDNA by the annealing procedure. No remarkable changes in size and distribution were confirmed before and after encapsulation of the ssDNA (Fig. 2(b)). On the other hand, the UCST of the microspheres decreased with increasing the quantity of encapsulated ssDNA increased (Fig. S2(b)). These results indicated that the interaction between the microspheres and the ssDNA was derived from the sulfobetaine unit in PEG-b-PDMAPS and phosphate in ssDNA. Here, the microspheres could not encapsulate the ssDNA below the UCST. The negative zeta potential of the
microsphere might affect electrostatic repulsion to ssDNA. Another possibility was that the sulfobetaine unit in the microspheres contributed to the formation of the microspheres and made it difficult to exchange from the sulfobetaine–sulfobetaine (dipole–dipole) interaction to the phosphate–sulfobetaine (ion–dipole) interaction. The microspheres encapsulated ssDNA exhibited both contact and separation, those behaviors were similar to that before encapsulation (Fig. S3). Therefore, the capability of exchanging ssDNA and the double strand formation in the microsphere were investigated by studying the contact of the ssDNA-encapsulating microspheres. We prepared 2 microsphere solutions to separately encapsulate complementary ssDNA. The DNA oligomers were fluorescently labeled and exhibited fluorescence resonance energy transfer (FRET) quenching if the double strand was formed. We chose TAMRA and BHQ-2 as a fluorophore and quencher, respectively, and the double strand formation resulted in a decreasing peak derived from TAMRA [24]. If the encapsulated ssDNA was transported and formed a double strand in the microsphere, the fluorescent intensity of TAMRA was reduced by FRET quenching. Fig. 3(a) shows monitoring the change of fluorescence intensity of TAMRA labeled ssDNA encapsulated in microsphere by injection of various solutions. Here, BHQ-2-labeled complementary ssDNA encapsulated in microspheres was co-cultured for 1 h in the solution. Water, NaCl solution, or 30-fold excess of complementary ssDNA solution were injected after the co-cultured microsphere solution and mixed under monitoring at 580 nm. The microspheres were immediately dissociated and the encapsulated ssDNA was released to form double strand with complementary ssDNA by injection of NaCl solution at the final concentration of 150 mM. Fig. 3(b) illustrated the behavior induced by addition of NaCl solution. To inhibit undesired FRET quenching by double strand formation in the presence of salt, large excess (×30) of non-labeled complementary ssDNA was added for the evaluation of double strand formation by trading ssDNA in the microsphere. In this system, TAMRA-labeled ssDNA in the microsphere was inhibited to form complex with the complementary BHQ-2-labeled ssDNA in solution. As the results, only double stranded DNA in microsphere was detected as the decrease of fluorescence intensity. Therefore, we used this assay system for further evaluation. When nonencapsulated complementary BHQ-2-labeled ssDNA was added to TAMRA-labeled ssDNA encapsulated microsphere solution, the fluorescent intensity of TAMRA was slightly decreased (
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Trading polymeric microspheres: Exchanging DNA molecules via microsphere interaction Nobuyuki Morimoto a,∗ , Kanna Muramatsu a , Shin-ichiro M. Nomura b , Makoto Suzuki a a
Department of Materials Processing, Graduate School of Engineering, Tohoku University, 6-6-02 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, 6-6-01 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan b
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 18 December 2014 Accepted 8 February 2015 Available online 17 February 2015 Keywords: Zwitterionic block copolymers Self-assembly Microspheres Single-stranded DNA oligomer Double strand formation
a b s t r a c t A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole–dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1 m) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3 -TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5 -BHQ-2labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the fundamental aspects of signal transduction and molecule delivery in cell–cell communication, contact-dependent signaling plays important roles, especially in immune and developmental systems and in the control of cell growth, differentiation, and morphogenesis [1]. The paracrine system and signaling via gap junctions are the major pathways of contact-dependent signaling. In paracrine signaling, the paracrine factors of signal molecules released from a cell diffuse locally to neighboring cells. Specific receptors exchange the signals to control the cell’s internal conditions. In contrast, signaling via gap junctions between contacted cytosols is facilitated diffusion. The gap junction intercellular channel regulates the transport of molecules by their molecular weight. In both types of signaling pathways, diffusion of signaling molecules is skillfully regulated. Biomimicking such contact-dependent transport system has led to the design of novel artificial microreactors, sensor devices and drug delivery systems. Polymeric microspheres, comparable in size to living cells, have been designed not only solid spheres but also hollow spheres
∗ Corresponding author. Tel.: +81 22 795 7365; fax: +81 22 795 7313. E-mail address: [email protected] (N. Morimoto). http://dx.doi.org/10.1016/j.colsurfb.2015.02.014 0927-7765/© 2015 Elsevier B.V. All rights reserved.
or vesicles [2–4], hydrogels [5,6], coacervates [7–10], and so on. There are many reports of controlling the release of encapsulated molecules in microspheres by simple diffusion or external stimuli [6,11–14]. However, the efficiency of the transport of encapsulated molecules between microspheres is expected to be low because of the simple, uncontrollable diffusion of the released molecules. In addition, the released molecules could be encapsulated into other microspheres under the same conditions with release in the absence of receptors. Therefore, construction of a contactdependent transport system would improve the effective transport of molecules between microspheres. Poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PDMAPS is known for its biocompatibility [15,16] and thermoresponsiveness of upper critical solution temperature (UCST) [17–19]. Those properties are derived from betaine structure of PDMAPS in the side chain. Below the UCST, the PDMAPS forms an unstable aggregate by the dipole–dipole interaction of the sulfobetaine units. Self-assembled nanoparticles have been designed by controlling the dipole–dipole interaction in water [20]. In our previous study, we prepared a block copolymer of poly(ethylene glycol)-b-PDMAPS (PEG-b-PDMAPS) via reversible addition fragmentation transfer (RAFT) polymerization [21]. The PEG-b-PDMAPS formed self-assembled and multi-layered microspheres in cold water. The PEG block improved the colloidal
N. Morimoto et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 94–99
stability of PDMAPS without losing the thermoresponsiveness. PEG-b-PDMAPS microsphere was controllable against dissociation above their UCST, as well as by the addition of salt. We report here the distinctive interaction between PEG-b-PDMAPS microspheres and the transport properties of encapsulated molecules using single-stranded DNA oligomers (ssDNA) as a model. 2. Experimental 2.1. Materials PEG-b-PDMAPS was prepared as reported previously [21]. The molecular weight of PEG-b-PDMAPS polymers were characterized by 1 H NMR and gel permeation chromatography (GPC) as shown in Table S1. 5 -Carboxymethylrhodamine (TAMRA) or 3 -Black Hole Quencher-2 (BHQ-2) modified DNA oligomers were purchased from FASMAC (Kanagawa, Japan). The sequences were as follows: TAMRA – 5 -TGT GGT ATG GCT GAT TAT GA-3 BHQ-2 – 3 -ACA CCA TAC CGA CTA ATA CT-5 The DNA oligomers were purified by high-performance liquid chromatography (HPLC) and analyzed by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectroscopy. Other reagents were purchased from Sigma–Aldrich (St. Louis, MO) and were used without further purification. 3. Methods
95
(IX-71, Olympus, Tokyo, Japan) equipped with a complementary metal–oxide–semiconductor (CMOS) camera (Neo 5.5 sCMOS, Andor, Northern Island, UK). 3.4. Evaluation of double strand formation Double strand formation of complementary ssDNA encapsulated in PEG-b-PDMAPS was evaluated as evidence of interaction between microspheres. Complementary ssDNA, one of modified fluorophore TAMRA in the 3 -terminus and the other of modified BHQ-2 in the 5 -terminus, were separately encapsulated. The ssDNA encapsulated microsphere solutions were mixed at r.t. after which the mixture was incubated at test temperature with a shaking rate of 60 min−1 . Double strand formation was evaluated by a decrease of the fluorescence intensity of TAMRA. The microsphere mixture was added to 150 mM (final concentration) NaCl solution containing 30 times excess (final concentration) of complementary ssDNA without TAMRA immobilization to inhibit unnecessary quenching. Fluorescence spectra were recorded with a fluorescence spectrometer (F-2500, Hitachi, Tokyo, Japan). The excitation wavelength was 547 nm, and spectra were recorded from 550 to 700 nm. The value of relative fluorescent intensity was calculated by the following equation: Relative fluorescent intensity =
I(t) − I(q) I(0) − I(q)
where I(0) is initial fluorescent intensity, I(t) is that at time t, and I(q) is that after quenching by addition of NaCl solution in the absence of excess complementary ssDNA.
3.1. Preparation of PEG-b-PDMAPS microspheres 4. Results and discussion PEG-b-PDMAPS (final concentration of sulfobetaine unit in polymer: 10 mM) was dispersed in pure water and the solution heated to 70 ◦ C. After 10 min, the solution was cooled to 20 ◦ C for 30 min. For encapsulating the DNA oligomers, DNA solution was mixed with polymer solution at r.t. and then the mixture was annealed by the same treatment as with the polymer solution. All DNA experiments except UV measurements were performed at 10 M phosphates in the ssDNA. 3.2. Characterization of microspheres The size and distribution of the PEG-b-PDMAPS microspheres were evaluated with a high performance laser diffraction analyzer (LA-950, HORIBA, Kyoto, Japan). The PEG-b-PDMAPS microsphere solution was dropped into the chamber cells in the proper scattering intensity. The solution was measured at 25 ◦ C. Zeta potential of PEG-b-PDMAPS microspheres was evaluated by an electrophoretic light scattering analyzer (SZ-100, HORIBA) using a disposable cell equipped with a carbon electrode. The temperature dependence of the cloud point in the PEG-b-PDMAPS microsphere solutions was monitored by UV–vis spectroscopy using a spectrometer (V-630, JASCO, Tokyo, Japan) equipped with a Peltier temperature control unit. The PEG-b-PDMAPS microsphere solution (10 mM in sulfobetaine unit) in the presence or absence of ssDNA was monitored at 550 nm under heating from 5 ◦ C to 65 ◦ C at a heating rate of 1 ◦ C min−1 . 3.3. Microscopic observation The microspheres were optically observed by use of a microscope. A drop of microsphere solution was covered by slide glass and lightly sealed. The specimen was observed and recorded with 10 fps at r.t. Fluorescence-labeled ssDNA encapsulated in PEG-bPDMAPS microspheres was observed by fluorescent microscope
4.1. Interaction between microspheres The characterization and solution properties of PEG-b-PDMAPS and the microspheres used in this study are shown in Table S1 and Table 1, respectively. PEG-b-PDMAPS was coded by the polymerization degrees of PEG and PDMAPS such as 110-73. First, narrow size distributed microspheres with a size of ∼1 m were confirmed in the 110 series of PEG. The longer PEG chain in PEG-b-PDMAPS was assumed to prevent aggregation and supply the colloidal stability of microsphere. The longer PEG chain increased the PEG composition of PEG-b-PDMAPS that increased the hydrophilicity of microsphere. Also the longer PEG-chain might work the excluded volume effect at the outer surface of microspheres. Another possibility was enhancing the phase separation effect between the longer PEG chain and PDMAPS chain in different PEG-b-PDMAPS. On the other hand, the size of 44-81 was not determined because the measured temperature was higher than the UCST. Fig. 1 shows time-lapse images of 110-73 microspheres dispersed in water at 25 ◦ C. Following the time course of the microsphere solution with steps of 0.1 s, contact, fusion and separation of the microspheres were observed in many places, as indicated by the arrows in Fig. 1. There were primarily three kinds of behaviors while the microspheres were in contact. (i) Two microspheres were fused, and then the fused microsphere was immediately (∼0.2 s) split into 2 microspheres Table 1 Solution properties of PEG-b-PDMAPS microspheres at 25 ◦ C. Polymer
Size [m]
Zeta-potential [mV]
Cloud point [◦ C]
22-74 44-81 110-61 110-73 110-119
2.30 ± 0.6 N.D. 0.94 ± 0.1 1.16 ± 0.1 0.78 ± 0.2
−27.9 −36.7 −33.1 −33.7 −36.8
48.5 23.6 37.0 42.0 28.9
± ± ± ± ±
3.4 1.6 4.4 1.8 0.6
± ± ± ± ±
1.7 3.3 2.9 1.7 2.9
96
N. Morimoto et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 94–99
Fig. 1. Microscopic observation of interaction between PEG-b-PDMAPS (110-73) microspheres in water at 25 ◦ C. The arrows indicated the contact of microspheres. Time 0: start of observation.
again with a budding-like behavior. In other cases, 2 microspheres were in contact and formed a peanut-like structure. Then, they (ii) rotated as a unit or (iii) rotated around another microsphere. In all cases, no remarkable size change was observed before and after the contact. The zeta potential of the microspheres was measured to analyze the contact behavior between the microspheres. Interestingly, all microspheres exhibited negative zeta potentials although the chemical structure of PEG-b-PDMAPS showed neutral (Table 1). These solutions exhibited neutral above (>10 ◦ C) the UCST or adding NaCl (>100 mM), when the microspheres were completely dissociated. Sulfobetaine side chain in a PDMAPS unit can form three types of cyclic conformations in water. These are: (1) formation between cationic and anionic groups in a monomer residue (intragroup), formation by head-to-tail stacking of the cationic and anionic groups, (2) within a single molecule (intrachain), or (3) between the neighboring macromolecules (interchain). These heterogenous combinations of betaine side chains might enable the microspheres to have a negative charge. Aside from the electrostatic repulsion, dipole–dipole interaction and van der Waals forces acted as attractive forces between microspheres. These attractive and repulsive forces were also intricately related to Brownian motion of microsphere. Moreover, PEG chain increased dispersion stability of microspheres, and also the multilayered structure of the microsphere might contribute to prevent from their fusion. These multiple factors would have caused the interesting interaction between microspheres. 4.2. Exchanging DNA molecules via microsphere interaction The unique behaviors of contact between microspheres were then applied to transport the encapsulated molecules in the microspheres. Here, we applied double strand formation by 2 kinds of microspheres those were separately encapsulated complementary single-stranded DNA oligomers (ssDNA, 20 mer). PDMAPS is known to interact with DNA [22,23]. First, we tried to encapsulate ssDNA into microspheres. PEG-b-PDMAPS microsphere solutions were heated to 70 ◦ C to dissociate microsphere, and then cooled to 20 ◦ C to form microsphere again. Fig. 2(a) shows a phase contrast microscopic image (upper) and fluorescent microscopic image (lower) of TAMRA-labeled ssDNA encapsulated in PEG-bPDMAPS (110-73) microspheres with a sulfobetaine (correspond to cation)/phosphate ([DMAPS]/[P]) ratio of 1000. No aggregation was confirmed by the encapsulation of ssDNA into microspheres. Though Brownian motion made difficult to observe, most of the microspheres were confirmed as having encapsulated TAMRAlabeled ssDNA by the annealing procedure. No remarkable changes in size and distribution were confirmed before and after encapsulation of the ssDNA (Fig. 2(b)). On the other hand, the UCST of the microspheres decreased with increasing the quantity of encapsulated ssDNA increased (Fig. S2(b)). These results indicated that the interaction between the microspheres and the ssDNA was derived from the sulfobetaine unit in PEG-b-PDMAPS and phosphate in ssDNA. Here, the microspheres could not encapsulate the ssDNA below the UCST. The negative zeta potential of the
microsphere might affect electrostatic repulsion to ssDNA. Another possibility was that the sulfobetaine unit in the microspheres contributed to the formation of the microspheres and made it difficult to exchange from the sulfobetaine–sulfobetaine (dipole–dipole) interaction to the phosphate–sulfobetaine (ion–dipole) interaction. The microspheres encapsulated ssDNA exhibited both contact and separation, those behaviors were similar to that before encapsulation (Fig. S3). Therefore, the capability of exchanging ssDNA and the double strand formation in the microsphere were investigated by studying the contact of the ssDNA-encapsulating microspheres. We prepared 2 microsphere solutions to separately encapsulate complementary ssDNA. The DNA oligomers were fluorescently labeled and exhibited fluorescence resonance energy transfer (FRET) quenching if the double strand was formed. We chose TAMRA and BHQ-2 as a fluorophore and quencher, respectively, and the double strand formation resulted in a decreasing peak derived from TAMRA [24]. If the encapsulated ssDNA was transported and formed a double strand in the microsphere, the fluorescent intensity of TAMRA was reduced by FRET quenching. Fig. 3(a) shows monitoring the change of fluorescence intensity of TAMRA labeled ssDNA encapsulated in microsphere by injection of various solutions. Here, BHQ-2-labeled complementary ssDNA encapsulated in microspheres was co-cultured for 1 h in the solution. Water, NaCl solution, or 30-fold excess of complementary ssDNA solution were injected after the co-cultured microsphere solution and mixed under monitoring at 580 nm. The microspheres were immediately dissociated and the encapsulated ssDNA was released to form double strand with complementary ssDNA by injection of NaCl solution at the final concentration of 150 mM. Fig. 3(b) illustrated the behavior induced by addition of NaCl solution. To inhibit undesired FRET quenching by double strand formation in the presence of salt, large excess (×30) of non-labeled complementary ssDNA was added for the evaluation of double strand formation by trading ssDNA in the microsphere. In this system, TAMRA-labeled ssDNA in the microsphere was inhibited to form complex with the complementary BHQ-2-labeled ssDNA in solution. As the results, only double stranded DNA in microsphere was detected as the decrease of fluorescence intensity. Therefore, we used this assay system for further evaluation. When nonencapsulated complementary BHQ-2-labeled ssDNA was added to TAMRA-labeled ssDNA encapsulated microsphere solution, the fluorescent intensity of TAMRA was slightly decreased (
