- Email: [email protected]
Preparation of multifunctional micelles from two different amphiphilic block copolymers
Colloids and Surfaces A 537 (2018) 566–571
Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Preparation of multifunctional micelles from two different amphiphilic block copolymers Junjira Tanuma, Uiyoung Hana, Jong wook Shinb, Jinkee Honga, a b
MARK
⁎
School of Chemical Engineering and Materials Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea Department of Internal Medicine, Chung-Ang University College and School of Medicine, Chung-Ang University Hospital, Seoul, Republic of Korea
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Micelle Block copolymer Multi-functional Core-shell pH sensitive
Micelles are widely used in drug delivery owing to their attractive properties such as controllable drug release rates and the ability to target certain locations by conjugating with specific molecules. However, with the current state of understanding, only the core–shell and corona sites of micelles can be utilized. To form a micelle, each block copolymer was dissolved in DMF and then dropped into a polar solvent. Polymer micelles were formed through the agglomeration of the hydrophobic part that constituted a non-polar core and a hydrophilic corona. In this study, poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP) and poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) formed micelles with a PS core in water. The hydrophobic PS domains were insoluble in the aqueous phase, which led to aggregation. Furthermore, from the pH-sensitive characteristic of weak polyelectrolyte, the changing of pH condition has an affected to the degree of ionization. Due to their characteristics, we prepared micelles from block copolymers at various pH values to increase the functionality of the micelles.
1. Introduction Block copolymer micelles (BCMs) are popular therapeutic delivering molecules due to their ability to assemble and disassemble under certain conditions, allowing encapsulation and release of therapeutic molecules in specific environments [1–3]. Using the self-assembling
⁎
ability of block copolymers in certain solvents, multicomponent nanostructures can be fabricated by incorporating particles in the core of BCMs [4,5]. Furthermore, BCMs combined with the layer-by-layer (LbL) assembly technique could allow the construction of layered structures of conjugates of BCMs and other molecules [6,7]. Usually, a variety of components such as therapeutic proteins, magnetic particles,
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Hong).
http://dx.doi.org/10.1016/j.colsurfa.2017.10.042 Received 30 August 2017; Received in revised form 18 October 2017; Accepted 19 October 2017 Available online 19 October 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
Colloids and Surfaces A 537 (2018) 566–571
J. Tanum et al.
water-soluble anticancer agents and decrease the release rate of paclitaxel (PTX), the latter was conjugated with oligo(L-lactic acid) (o(LA)) and loaded into the PEG-b-PLA micelles, which displayed enhanced compatibility compared to that of unconjugated PTX. Further, on o(LA) conjugation, the amount of PTX loading increased from 11 to 54% [19]. Through the encapsulation and conjugation of BCM with other molecules, one can utilize the corona and core sites to improve the chemical and/or physical properties of the therapeutic micelle carriers. For the past ten years, micelles have been used as containers for delivering many kinds of molecules. Their loading, release, and targeting properties have steadily been improved. However, micelles prepared from a single block copolymer might limit the range of molecules that can be encapsulated. Hence, researchers have tried using multiple block copolymer-based, multifunctional micelles to incorporate different components [20,21]. In this study, highly functional micelles were formed from two different block copolymers with different charges, poly(styrene)-blockpoly(acrylic acid) (PS-b-PAA) and poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP), as schematically shown in Fig. 1. The block copolymer solution was prepared by completely dissolving PS-b-PAA and PSb-P4VP in DMF. Then, the solution was dropped into water (polar solvent) to form a mixed micelle. The micelle prepared at pH 4.63 is expected to consist of a PS core with PAA and P4VP corona. Using the pH-
and drugs are encapsulated and conjugated within BCMs through electrostatic interactions [8–11]. The BCM drug carriers effectively combine with other compounds to form multilayered films, enabling a well-controlled drug release [6,12]. By using external stimuli such as pH, temperature, and light, release and encapsulation of drugs with BCMs can be controlled [13–15]. For example, polymer micelles from poly(2-(diisopropylamino)ethyl methacrylate) and poly(2-(dibutylamino)ethyl methacrylate) were used in the gastrointestinal (GI) tract, since both the polymers dissolve in acidic solution and flocculate in alkaline solution, thereby responding to pH changes in the GI tract [16]. Polyethylene glycol (PEG) conjugated with 20% chlorin e6 (Ce 6) was used to prepare nanomicelles; the outer part of the micelles chelated with Cu2+ and used as an optical imaging agent in organic photodynamic therapy (PDT) [9]. Furthermore, a combination of monophosphoryl lipid A encapsulated within a poly(ethylene glycol)-blockpoly(propylenesulfide) (PEG-b-PPS) block copolymer exhibited a role in cellular and humoral response [17]. Nowadays, the research on BCMs not only focuses on developing new methods and applications, but also on improving the properties of micelles, such as their drug loading efficiency, to achieve better therapeutic results. For example, cancer treatment was improved using poly (ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) micelles loaded with multiple anticancer agents [18]. To deliver sparingly
Fig 1. Scheme of micelle formation with two different block copolymers. PS-b-PAA and PS-b-P4VP were dissolved in DMF and assembled into micelles in DI water at pH 4.63.
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2.2. Fabrication of micelles
Table 1 Average charge of the micelles made of two different block copolymers at various preparation pH values. pH
Zeta-potential (mV)
3.41 4.63 6.85 10.4
35.6 −42 to 29 −65 −73.7
Due to the well-dissolve of block copolymers in DMF solution. PS-bPAA and PS-b-P4VP block copolymers were dissolved in DMF at a concentration of 0.5 mg/mL. Then stirred for 1 h to achieve complete dispersion in DMF. The mixed block copolymer solution was filtered using a 0.45 μm mesh size filter and added dropwise to 50 mL of distilled water (DI) under vigorous stirring. The pH of the DI water was varied at this step (vide infra). The solution was stirred overnight at room temperature to complete the micelle formation and was dialyzed against water with a 14 kDa cut-off membrane for 2 d. After dialysis, micelles were obtained after centrifugation at 5000 rpm for 10 min. After the larger aggregates formed sediment at the bottom, the supernatant solution was characterized.
Table 2 Average size measure by DLS of PS-b-PAA, PS-b-P4VP and PS-bPAA/P4VP micelles prepared at pH4.63 (n = 6). Micelle
size by DLS (nm)
PS-b-PAA PS-b-P4VP PS-b-PAA/P4VP
154.5 ± 20.3 113.9 ± 5.3 578.9 ± 147.9
2.3. Characterization To determine the micelle size and morphology using scanning electron microscopy (SEM), polyacrylic acid (PAA) (1 mg/mL solution, pH 3.5) and poly(allylamine hydrochloride) (PAH) (1 mg/mL solution, pH 7.5) were used as buffering layers. (PAA/PAH) films with 9.5 bilayers ((PAA/PAH)9.5) were fabricated on a Si wafer using a dippingLbL assembly technique. First, the Si wafer was treated with O2 plasma for 2 min and then dipped in PAH solution for 10 min. The Si wafer was then washed twice by dipping in DI water for 2 min each time. Then, PAA was deposited onto the PAH layer by the same method. Finally, micelles made of the two block copolymers were deposited on top of the (PAA/PAH)9.5 multilayer films. Field-emission scanning electron microscopy (FE-SEM, LIBRA 120 microscope, Carl Zeiss) was used to determine the micelle size and morphology. The SEM images were obtained at an acceleration voltage of 5 kV. The morphology and size of the micelles were also studied by atomic force microscopy (AFM, NX10, Park Systems) in non-contact mode. The zeta potential and size of the micelles were measured with a nanoparticle analyzer model SZ-100 (Horiba) and Fourier transform infrared spectroscopy (FTIR) was performed using a FTIR 4700 (JASCO) instrument.
dependent properties of weak polyelectrolytes, we can control the formation of block copolymer micelles by controlling the pH [22]. Because of the assembly of two different block copolymers, these BCMs has a potential to interact in multiple ways at the corona site and a variety of mechanisms are possible at the core and corona of each micelle.
2. Materials and methods 2.1. Materials PS4.3k-b-PAA19.5k and PS3.3k-b-P4VP18.7k block copolymers were obtained from Polymer Source, Inc. N,N-Dimethylformamide (DMF) was purchased from SAMCHUN. Hydrochloric acid and sodium hydroxide were obtained from Daejung.
Fig. 2. FE-SEM image showing the morphology of the micelles prepared with two different block copolymers at pH 4.63.
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deprotonated and becomes insoluble in water, while the PAA segment is protonated. Therefore, PS-b-PAA assembles into micelles at pH 6.85 showing a negative charge in the zeta potential. Under highly basic conditions, the PAA fragment shows a high degree of ionization. As a result, no micelle formation occurs at this pH and a very clear solution with agglomerates of PS-b-P4VP block copolymer is obtained. The average charge of the mixed block copolymer micelles at various pH values using zeta potential analysis is summarized in Table 1. The results show that, upon assembly, the PS-b-PAA/P4VP micelles still possess the typical characteristics of weak polyelectrolytes, as their charge density depends on the pH. pH 4.63 is the best condition for the formation of PS-b-PAA/P4VP micelles. PS-b-PAA/P4VP micelles were prepared and characterized using DLS, SEM, AFM, zeta-potential, and FTIR. The size of the PS-b-PAA/ P4VP micelles prepared at pH 4.63 compared with that of PS-b-PAA and PS-b-P4VP micelle is shown in Table 2. The average size of three micelles after preparation was measured at pH 4.63. The sizes determined using dynamic light scattering (DLS) were 154.5, 113.9, and 578.9 for PS-b-PAA, PS-b-P4VP, and PS-b-PAA/P4VP micelles, respectively. The negative and positive charge segment contained in the micelle causes agglomeration and makes the micelle bigger. Renata et al. showed that the size of the blended micelle was larger than that of the unmixed block copolymer micelle, because of the pairing of the PAA and P4VP strands. These paired strands are expected to be hydrophobic, which aggregate to form a hydrophobic shell around the PS core. Thus, in our study, we expect the mixed micelle size in the solution to be similar to that of the PS-b-PAA micelle. Due to the aggregation of the mixed micelles, size could not be measured using DLS. SEM was used to observe the size and morphology of the PS-b-PAA/P4VP micelles. The SEM image (Fig. 2) shows that the PS-b-PAA/P4VP micelles synthesized at pH 4.63 are spherical. The PS-b-PAA/P4VP micelles are more agglomerated than the corresponding homogeneous micelles. These micelles undergo sedimentation after a few days because of the electrostatic interaction of opposite charges in the corona. Owing to the different percentage of ionization of the PAA and P4VP groups, the micelles display a range of surface charges depending on the solution pH. The
Fig. 3. PS-b-PAA/P4VP micelle size distribution obtained from SEM image (SD, n = 4).
3. Results and discussion Micelles from two different block copolymers were synthesized using PS-b-PAA and PS-b-P4VP, denoted as PS-b-PAA/P4VP. The pH of the solution was optimized for micelle formation. Previously, it was reported that, at pH < 5, PS-b-P4VP micelles form with a PS core and a P4VP shell. Above pH 5.5, PS-b-P4VP deprotonation makes it waterinsoluble, but at pH > 3, PS-b-PAA forms micelles due to protonation of the carboxylic groups in PAA [23]. In our study, at pH 3.4, the zeta potential analysis revealed a positive charge due to PS-b-P4VP micelle formation and upon stirring overnight, a clear solution with small aggregates of PS-b-P4VP micelles was obtained (Fig. S1 in the Supplement material). At pH 4.63, both P4VP and PAA are protonated and formed micelles in the solution having zeta potentials different from the individual PAA and P4VP micelles. At pH 6.85 and 10.4, P4VP is
Fig. 4. AFM image of the micelles prepared with two different block copolymers at pH 4.63.
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site and that the insoluble P4VP part deposited on the PS core in aqueous solution at room temperature [26]. In the dry state, PAA corona collapses and makes the micelle size similar to that of the P4VP part. Thus, we can predict the formation of complex micelles by comparing their size with that of the pure block copolymer micelles. For surface charge measurement, each micelle was prepared in their individual condition. To prevent agglomeration during micelle assembly, PS-b-PAA was prepared at pH 10, PS-b-P4VP micelle at pH 3, and PS-b-PAA/P4VP micelle at pH 4.63. After preparation and dialysis, every micelle was diluted with deionized water at various pH. The zetapotential of the micelle at various pH values is shown in Fig. 5. The surface charge of micelles is known to depend on pH [13,16,22,23,26]. Therefore, we measured the surface charge of PS-b-PAA, PS-b-P4VP, and PS-b-PAA/P4VP micelles at pH values ranging from pH 2.5–10. For the PS-b-PAA micelle, PAA is neutral at low pH. On increasing the pH, the charge on the corona becomes stronger due to protonation of the carboxylic group [23]. However, P4VP is highly charged at low pH due to protonation of the pyridine group. At pH > 5.5, PS-b-P4VP has no charge. Surface charge comparison shows that PS-b-PAA/P4VP micelles show the characteristics of both PS-b-PAA and PS-b-P4VP, with positive charge at low pH and negative charge at high pH (Fig. 6). At low pH, the PS-b-PAA/P4VP micelle has positive charge due to deprotonation of PAA and it becomes neutral at this pH. At pH 4.6, the zeta-potential is 1.6 mV due to the presence of both positive and negative charges in this micelle. PAA and P4VP form a hydrophobic ladder pair with neutral charge, while the PAA or P4VP segment that remains in the solvent results in a residual charge. Under basic conditions, the P4VP segment deprotonates, making the micelle negatively charged. The FTIR spectra of the PS-b-PAA micelle show the characteristic peak of carboxylic group at 1715 cm−1 and PS-b-P4VP micelle shows a pyridine ring peak at 1598 cm−1 (data was shown in Fig. S2, Supplement material). Compared to the PS-b-PAA/P4VP micelle, the complex micelle shows the characteristic peak of both carboxylic group and pyridine ring. However, the peaks shifted to higher wavenumbers owing to the interaction between the pyridine and carboxylic groups [27,28]. BCMs formed by the self-assembly of PS-b-PAA and PS-b-P4VP
Fig. 5. The zeta-potential value of PS-b-PAA (■), PS-b-P4VP ( ), and PS-b-PAA/P4VP ( ) micelle in different pH condition (SD, n = 6).
AFM image (Fig. 4) was used to confirm the morphology of the mixed micelle. The result clearly shows the aggregation of PS-b-PAA/P4VP micelles, while homogeneous micelles exhibit good dispersion on the surface [24,25]. Fig. 3 shows the size distribution of the PS-b-PAA/P4VP micelles calculated from the SEM image. In micelle size distribution plot, we collect the data from 4 SEM figure in different area. After that, the average from 4 pictures was used to calculate the standard deviation (SD) of the micelle size distribution. The size distribution was broad with the maximum population in the range of 60–70 nm. However, when considering the size of the PS-b-PAA/P4VP micelle, the size should be similar to that of the PS-b-P4VP micelle. As reported in the literature, the corona size of complex micelles can be estimated as the length of the shorter chain in the combination [22]. Shi et al. reported that the polymer micelle contained both PAA and P4VP at the corona
Fig. 6. The morphology of PS-b-PAA/P4VP micelle at different pH condition (n = 6).
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block copolymers have a hydrophobic core and a hydrophilic shell. We envision that these micelles might have great potential for incorporating a variety of molecules, because of the presence of both negative and positive charges in the corona.
[7]
[8]
4. Conclusions [9]
Micelles prepared from two different block copolymers can improve the efficiency of drug delivery. Due to their improved functionality, a large number of therapeutic drugs can be loaded into the micelles. PS3.5k-b-PAA23k and PS3.3k-b-P4VP18.7k block copolymers were assembled into different micelles at various pH values. The zeta potential data revealed that, at pH 3.41, only PS-b-P4VP micelles are formed. At pH 4.63, both the individual and mixed micelles of PS-b-P4VP and PS-bPAA are formed. At pH > 6, the PAA segment dominates and PS-bPAA micelles are formed. However, PS-b-PAA cannot assemble into micelles under highly alkaline conditions because of its highly charged surface.
[10] [11]
[12] [13]
[14]
[15]
Conflict of interest The authors declare that they have no conflict of interest.
[16]
Acknowledgements
[17]
This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIT [2016M3A9C6917405]. Additionally, this research was supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C-3266, HI15C-1653). Also this research is supported by "The Project of Conversion by the Past R & D Results" through the Ministry of Trade, Industry and Energy (MOTIE) and the Korea Institute for Advancement of Technology (KIAT) (N0002496, 2017).
[18]
[19]
[20] [21]
[22]
Appendix A. Supplementary data [23]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2017.10.042.
[24]
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Preparation of multifunctional micelles from two different amphiphilic block copolymers Junjira Tanuma, Uiyoung Hana, Jong wook Shinb, Jinkee Honga, a b
MARK
⁎
School of Chemical Engineering and Materials Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea Department of Internal Medicine, Chung-Ang University College and School of Medicine, Chung-Ang University Hospital, Seoul, Republic of Korea
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Micelle Block copolymer Multi-functional Core-shell pH sensitive
Micelles are widely used in drug delivery owing to their attractive properties such as controllable drug release rates and the ability to target certain locations by conjugating with specific molecules. However, with the current state of understanding, only the core–shell and corona sites of micelles can be utilized. To form a micelle, each block copolymer was dissolved in DMF and then dropped into a polar solvent. Polymer micelles were formed through the agglomeration of the hydrophobic part that constituted a non-polar core and a hydrophilic corona. In this study, poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP) and poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) formed micelles with a PS core in water. The hydrophobic PS domains were insoluble in the aqueous phase, which led to aggregation. Furthermore, from the pH-sensitive characteristic of weak polyelectrolyte, the changing of pH condition has an affected to the degree of ionization. Due to their characteristics, we prepared micelles from block copolymers at various pH values to increase the functionality of the micelles.
1. Introduction Block copolymer micelles (BCMs) are popular therapeutic delivering molecules due to their ability to assemble and disassemble under certain conditions, allowing encapsulation and release of therapeutic molecules in specific environments [1–3]. Using the self-assembling
⁎
ability of block copolymers in certain solvents, multicomponent nanostructures can be fabricated by incorporating particles in the core of BCMs [4,5]. Furthermore, BCMs combined with the layer-by-layer (LbL) assembly technique could allow the construction of layered structures of conjugates of BCMs and other molecules [6,7]. Usually, a variety of components such as therapeutic proteins, magnetic particles,
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Hong).
http://dx.doi.org/10.1016/j.colsurfa.2017.10.042 Received 30 August 2017; Received in revised form 18 October 2017; Accepted 19 October 2017 Available online 19 October 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
Colloids and Surfaces A 537 (2018) 566–571
J. Tanum et al.
water-soluble anticancer agents and decrease the release rate of paclitaxel (PTX), the latter was conjugated with oligo(L-lactic acid) (o(LA)) and loaded into the PEG-b-PLA micelles, which displayed enhanced compatibility compared to that of unconjugated PTX. Further, on o(LA) conjugation, the amount of PTX loading increased from 11 to 54% [19]. Through the encapsulation and conjugation of BCM with other molecules, one can utilize the corona and core sites to improve the chemical and/or physical properties of the therapeutic micelle carriers. For the past ten years, micelles have been used as containers for delivering many kinds of molecules. Their loading, release, and targeting properties have steadily been improved. However, micelles prepared from a single block copolymer might limit the range of molecules that can be encapsulated. Hence, researchers have tried using multiple block copolymer-based, multifunctional micelles to incorporate different components [20,21]. In this study, highly functional micelles were formed from two different block copolymers with different charges, poly(styrene)-blockpoly(acrylic acid) (PS-b-PAA) and poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP), as schematically shown in Fig. 1. The block copolymer solution was prepared by completely dissolving PS-b-PAA and PSb-P4VP in DMF. Then, the solution was dropped into water (polar solvent) to form a mixed micelle. The micelle prepared at pH 4.63 is expected to consist of a PS core with PAA and P4VP corona. Using the pH-
and drugs are encapsulated and conjugated within BCMs through electrostatic interactions [8–11]. The BCM drug carriers effectively combine with other compounds to form multilayered films, enabling a well-controlled drug release [6,12]. By using external stimuli such as pH, temperature, and light, release and encapsulation of drugs with BCMs can be controlled [13–15]. For example, polymer micelles from poly(2-(diisopropylamino)ethyl methacrylate) and poly(2-(dibutylamino)ethyl methacrylate) were used in the gastrointestinal (GI) tract, since both the polymers dissolve in acidic solution and flocculate in alkaline solution, thereby responding to pH changes in the GI tract [16]. Polyethylene glycol (PEG) conjugated with 20% chlorin e6 (Ce 6) was used to prepare nanomicelles; the outer part of the micelles chelated with Cu2+ and used as an optical imaging agent in organic photodynamic therapy (PDT) [9]. Furthermore, a combination of monophosphoryl lipid A encapsulated within a poly(ethylene glycol)-blockpoly(propylenesulfide) (PEG-b-PPS) block copolymer exhibited a role in cellular and humoral response [17]. Nowadays, the research on BCMs not only focuses on developing new methods and applications, but also on improving the properties of micelles, such as their drug loading efficiency, to achieve better therapeutic results. For example, cancer treatment was improved using poly (ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) micelles loaded with multiple anticancer agents [18]. To deliver sparingly
Fig 1. Scheme of micelle formation with two different block copolymers. PS-b-PAA and PS-b-P4VP were dissolved in DMF and assembled into micelles in DI water at pH 4.63.
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2.2. Fabrication of micelles
Table 1 Average charge of the micelles made of two different block copolymers at various preparation pH values. pH
Zeta-potential (mV)
3.41 4.63 6.85 10.4
35.6 −42 to 29 −65 −73.7
Due to the well-dissolve of block copolymers in DMF solution. PS-bPAA and PS-b-P4VP block copolymers were dissolved in DMF at a concentration of 0.5 mg/mL. Then stirred for 1 h to achieve complete dispersion in DMF. The mixed block copolymer solution was filtered using a 0.45 μm mesh size filter and added dropwise to 50 mL of distilled water (DI) under vigorous stirring. The pH of the DI water was varied at this step (vide infra). The solution was stirred overnight at room temperature to complete the micelle formation and was dialyzed against water with a 14 kDa cut-off membrane for 2 d. After dialysis, micelles were obtained after centrifugation at 5000 rpm for 10 min. After the larger aggregates formed sediment at the bottom, the supernatant solution was characterized.
Table 2 Average size measure by DLS of PS-b-PAA, PS-b-P4VP and PS-bPAA/P4VP micelles prepared at pH4.63 (n = 6). Micelle
size by DLS (nm)
PS-b-PAA PS-b-P4VP PS-b-PAA/P4VP
154.5 ± 20.3 113.9 ± 5.3 578.9 ± 147.9
2.3. Characterization To determine the micelle size and morphology using scanning electron microscopy (SEM), polyacrylic acid (PAA) (1 mg/mL solution, pH 3.5) and poly(allylamine hydrochloride) (PAH) (1 mg/mL solution, pH 7.5) were used as buffering layers. (PAA/PAH) films with 9.5 bilayers ((PAA/PAH)9.5) were fabricated on a Si wafer using a dippingLbL assembly technique. First, the Si wafer was treated with O2 plasma for 2 min and then dipped in PAH solution for 10 min. The Si wafer was then washed twice by dipping in DI water for 2 min each time. Then, PAA was deposited onto the PAH layer by the same method. Finally, micelles made of the two block copolymers were deposited on top of the (PAA/PAH)9.5 multilayer films. Field-emission scanning electron microscopy (FE-SEM, LIBRA 120 microscope, Carl Zeiss) was used to determine the micelle size and morphology. The SEM images were obtained at an acceleration voltage of 5 kV. The morphology and size of the micelles were also studied by atomic force microscopy (AFM, NX10, Park Systems) in non-contact mode. The zeta potential and size of the micelles were measured with a nanoparticle analyzer model SZ-100 (Horiba) and Fourier transform infrared spectroscopy (FTIR) was performed using a FTIR 4700 (JASCO) instrument.
dependent properties of weak polyelectrolytes, we can control the formation of block copolymer micelles by controlling the pH [22]. Because of the assembly of two different block copolymers, these BCMs has a potential to interact in multiple ways at the corona site and a variety of mechanisms are possible at the core and corona of each micelle.
2. Materials and methods 2.1. Materials PS4.3k-b-PAA19.5k and PS3.3k-b-P4VP18.7k block copolymers were obtained from Polymer Source, Inc. N,N-Dimethylformamide (DMF) was purchased from SAMCHUN. Hydrochloric acid and sodium hydroxide were obtained from Daejung.
Fig. 2. FE-SEM image showing the morphology of the micelles prepared with two different block copolymers at pH 4.63.
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deprotonated and becomes insoluble in water, while the PAA segment is protonated. Therefore, PS-b-PAA assembles into micelles at pH 6.85 showing a negative charge in the zeta potential. Under highly basic conditions, the PAA fragment shows a high degree of ionization. As a result, no micelle formation occurs at this pH and a very clear solution with agglomerates of PS-b-P4VP block copolymer is obtained. The average charge of the mixed block copolymer micelles at various pH values using zeta potential analysis is summarized in Table 1. The results show that, upon assembly, the PS-b-PAA/P4VP micelles still possess the typical characteristics of weak polyelectrolytes, as their charge density depends on the pH. pH 4.63 is the best condition for the formation of PS-b-PAA/P4VP micelles. PS-b-PAA/P4VP micelles were prepared and characterized using DLS, SEM, AFM, zeta-potential, and FTIR. The size of the PS-b-PAA/ P4VP micelles prepared at pH 4.63 compared with that of PS-b-PAA and PS-b-P4VP micelle is shown in Table 2. The average size of three micelles after preparation was measured at pH 4.63. The sizes determined using dynamic light scattering (DLS) were 154.5, 113.9, and 578.9 for PS-b-PAA, PS-b-P4VP, and PS-b-PAA/P4VP micelles, respectively. The negative and positive charge segment contained in the micelle causes agglomeration and makes the micelle bigger. Renata et al. showed that the size of the blended micelle was larger than that of the unmixed block copolymer micelle, because of the pairing of the PAA and P4VP strands. These paired strands are expected to be hydrophobic, which aggregate to form a hydrophobic shell around the PS core. Thus, in our study, we expect the mixed micelle size in the solution to be similar to that of the PS-b-PAA micelle. Due to the aggregation of the mixed micelles, size could not be measured using DLS. SEM was used to observe the size and morphology of the PS-b-PAA/P4VP micelles. The SEM image (Fig. 2) shows that the PS-b-PAA/P4VP micelles synthesized at pH 4.63 are spherical. The PS-b-PAA/P4VP micelles are more agglomerated than the corresponding homogeneous micelles. These micelles undergo sedimentation after a few days because of the electrostatic interaction of opposite charges in the corona. Owing to the different percentage of ionization of the PAA and P4VP groups, the micelles display a range of surface charges depending on the solution pH. The
Fig. 3. PS-b-PAA/P4VP micelle size distribution obtained from SEM image (SD, n = 4).
3. Results and discussion Micelles from two different block copolymers were synthesized using PS-b-PAA and PS-b-P4VP, denoted as PS-b-PAA/P4VP. The pH of the solution was optimized for micelle formation. Previously, it was reported that, at pH < 5, PS-b-P4VP micelles form with a PS core and a P4VP shell. Above pH 5.5, PS-b-P4VP deprotonation makes it waterinsoluble, but at pH > 3, PS-b-PAA forms micelles due to protonation of the carboxylic groups in PAA [23]. In our study, at pH 3.4, the zeta potential analysis revealed a positive charge due to PS-b-P4VP micelle formation and upon stirring overnight, a clear solution with small aggregates of PS-b-P4VP micelles was obtained (Fig. S1 in the Supplement material). At pH 4.63, both P4VP and PAA are protonated and formed micelles in the solution having zeta potentials different from the individual PAA and P4VP micelles. At pH 6.85 and 10.4, P4VP is
Fig. 4. AFM image of the micelles prepared with two different block copolymers at pH 4.63.
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site and that the insoluble P4VP part deposited on the PS core in aqueous solution at room temperature [26]. In the dry state, PAA corona collapses and makes the micelle size similar to that of the P4VP part. Thus, we can predict the formation of complex micelles by comparing their size with that of the pure block copolymer micelles. For surface charge measurement, each micelle was prepared in their individual condition. To prevent agglomeration during micelle assembly, PS-b-PAA was prepared at pH 10, PS-b-P4VP micelle at pH 3, and PS-b-PAA/P4VP micelle at pH 4.63. After preparation and dialysis, every micelle was diluted with deionized water at various pH. The zetapotential of the micelle at various pH values is shown in Fig. 5. The surface charge of micelles is known to depend on pH [13,16,22,23,26]. Therefore, we measured the surface charge of PS-b-PAA, PS-b-P4VP, and PS-b-PAA/P4VP micelles at pH values ranging from pH 2.5–10. For the PS-b-PAA micelle, PAA is neutral at low pH. On increasing the pH, the charge on the corona becomes stronger due to protonation of the carboxylic group [23]. However, P4VP is highly charged at low pH due to protonation of the pyridine group. At pH > 5.5, PS-b-P4VP has no charge. Surface charge comparison shows that PS-b-PAA/P4VP micelles show the characteristics of both PS-b-PAA and PS-b-P4VP, with positive charge at low pH and negative charge at high pH (Fig. 6). At low pH, the PS-b-PAA/P4VP micelle has positive charge due to deprotonation of PAA and it becomes neutral at this pH. At pH 4.6, the zeta-potential is 1.6 mV due to the presence of both positive and negative charges in this micelle. PAA and P4VP form a hydrophobic ladder pair with neutral charge, while the PAA or P4VP segment that remains in the solvent results in a residual charge. Under basic conditions, the P4VP segment deprotonates, making the micelle negatively charged. The FTIR spectra of the PS-b-PAA micelle show the characteristic peak of carboxylic group at 1715 cm−1 and PS-b-P4VP micelle shows a pyridine ring peak at 1598 cm−1 (data was shown in Fig. S2, Supplement material). Compared to the PS-b-PAA/P4VP micelle, the complex micelle shows the characteristic peak of both carboxylic group and pyridine ring. However, the peaks shifted to higher wavenumbers owing to the interaction between the pyridine and carboxylic groups [27,28]. BCMs formed by the self-assembly of PS-b-PAA and PS-b-P4VP
Fig. 5. The zeta-potential value of PS-b-PAA (■), PS-b-P4VP ( ), and PS-b-PAA/P4VP ( ) micelle in different pH condition (SD, n = 6).
AFM image (Fig. 4) was used to confirm the morphology of the mixed micelle. The result clearly shows the aggregation of PS-b-PAA/P4VP micelles, while homogeneous micelles exhibit good dispersion on the surface [24,25]. Fig. 3 shows the size distribution of the PS-b-PAA/P4VP micelles calculated from the SEM image. In micelle size distribution plot, we collect the data from 4 SEM figure in different area. After that, the average from 4 pictures was used to calculate the standard deviation (SD) of the micelle size distribution. The size distribution was broad with the maximum population in the range of 60–70 nm. However, when considering the size of the PS-b-PAA/P4VP micelle, the size should be similar to that of the PS-b-P4VP micelle. As reported in the literature, the corona size of complex micelles can be estimated as the length of the shorter chain in the combination [22]. Shi et al. reported that the polymer micelle contained both PAA and P4VP at the corona
Fig. 6. The morphology of PS-b-PAA/P4VP micelle at different pH condition (n = 6).
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block copolymers have a hydrophobic core and a hydrophilic shell. We envision that these micelles might have great potential for incorporating a variety of molecules, because of the presence of both negative and positive charges in the corona.
[7]
[8]
4. Conclusions [9]
Micelles prepared from two different block copolymers can improve the efficiency of drug delivery. Due to their improved functionality, a large number of therapeutic drugs can be loaded into the micelles. PS3.5k-b-PAA23k and PS3.3k-b-P4VP18.7k block copolymers were assembled into different micelles at various pH values. The zeta potential data revealed that, at pH 3.41, only PS-b-P4VP micelles are formed. At pH 4.63, both the individual and mixed micelles of PS-b-P4VP and PS-bPAA are formed. At pH > 6, the PAA segment dominates and PS-bPAA micelles are formed. However, PS-b-PAA cannot assemble into micelles under highly alkaline conditions because of its highly charged surface.
[10] [11]
[12] [13]
[14]
[15]
Conflict of interest The authors declare that they have no conflict of interest.
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Acknowledgements
[17]
This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIT [2016M3A9C6917405]. Additionally, this research was supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C-3266, HI15C-1653). Also this research is supported by "The Project of Conversion by the Past R & D Results" through the Ministry of Trade, Industry and Energy (MOTIE) and the Korea Institute for Advancement of Technology (KIAT) (N0002496, 2017).
[18]
[19]
[20] [21]
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Appendix A. Supplementary data [23]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2017.10.042.
[24]
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