Surface and Aggregation Behavior of Pentablock Copolymer PNIPAM

Article pubs.acs.org/JPCB

Surface and Aggregation Behavior of Pentablock Copolymer PNIPAM7‑F127-PNIPAM7 in Aqueous Solutions P. Parekh,† S. Ohno,§ S. Yusa,§ Emílio V. Lage,∥ Matilde Casas,∥ I. Sández-Macho,∥ V. K. Aswal,‡ and P. Bahadur*,† †

Chemistry Department, V.N.S.G. University, Surat 395007, India Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400085, India § Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo, Japan ∥ Departamento de Química Física, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ‡

ABSTRACT: The triblock Pluronic F127 was modified by introducing poly(N-isopropylacrylamide) (PNIPAM) at both the poly(ethylene oxide) ends, and the pentablock copolymer soprepared was characterized by gel permeation chromatography and 1H NMR. The degree of polymerization of NIPAM blocks at the two ends was 7. The solution behavior and microstructure of copolymer aggregates in water and aqueous salt solution were examined and compared with F127 by UV−visible absorption spectroscopy, microdifferential scanning calorimetry, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The behavior of the pentablock copolymer at the air/water interface was determined by Langmuir film balance. Two lower critical solution temperatures were observed for pentablock copolymer, corresponding to poly(propylene oxide) and PNIPAM blocks, respectively. DLS studies show that micelle size increased with increase in temperature and in the presence of salt. SANS measurements provided temperaturedependent structural evolution of copolymer micelles in water and salt solution. The copolymer displays an isotherm with four classical regions (pancake, mushroom, brush, and condensed state). The study has potential applications in controlled drug delivery due to the tunable phase behavior and biocompatibility of the copolymer.

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INTRODUCTION

Different strategies have been employed to circumvent this problem of micelle disintegration on dilution. Mixtures of hydrophilic and hydrophobic pluronics is one approach, and such mixed micelles have been characterized.4,5 In other cases, cross-linking of micelle core using benzoyl peroxide,6 introduction of vegetable oil to increase hydrophobic interactions within the core7,8 or formation of hydrogel inside the micelle core (Plurogel),9 and stabilization of pluronic micelles by UV-light to form interpenetrating network of poly(pentaerythritol tetraacrylate)10 have been used. Few reports also exist on the synthesis of pentablock copolymers made of pluronics with attached polymer size blocks at the end of each PEO block. Such block copolymers with poly(N-isopropylacrylamide) (PNIPAM),11,12 and poly(lactide-co-glycolide),13 poly(amine methacrylate),14 and poly[N,N-(diethyl amino)ethyl methacrylate]15 have been synthesized. Lu et al.11 using PNIPAM-P123-PNIPAM found that lower critical solution temperature (LCST) of PPO block shifted from 24.4 to 29.0 °C when the degree of polymerization (DP) of PNIPAM block increased from 10 to 97. The LCST of PNIPAM is around 34.5 to 35.3 °C and less dependent on the block length. The terpolymers formed “associate” structures

Poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO−PPO−PEO) are commercially available amphiphilic triblock copolymers sold under the trade name Pluronic or Poloxamer. These copolymers have developed interest for their possible applications as nanoreservoirs for drug and gene delivery due to their nontoxicity and low immune response.1 PEO−PPO−PEO molecules self-assemble in aqueous solution and form core−shell micelles (ca. 10−30 nm); the micelle formation is highly temperature-dependent due to the heat-responsive middle PPO block. These micelles are of biomedical interest because PEO shell can resist protein adsorption, load an adequate amount of hydrophobic drug, organize into thermoreversible gels, and contain hydroxyl end groups to which receptor-specific ligands can be attached.2,3 However, these polymeric micelles are associated with a drawback of getting disintegrated into unimers upon dilution under physiological conditions that limits their direct use. The stability of copolymer micelles largely depends on the hydrophobicity/hydrophilicity of copolymer (very hydrophobic copolymers like L121) due to their low cloud point (CP) and do not micellize and remain as unstable vesicles in water. Similarly, hydrophilic copolymer like F127 forms less stable micelles compared with more hydrophobic copolymer P123. © 2016 American Chemical Society

Received: April 19, 2016 Revised: June 29, 2016 Published: July 6, 2016 7569

DOI: 10.1021/acs.jpcb.6b03948 J. Phys. Chem. B 2016, 120, 7569−7578

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The Journal of Physical Chemistry B

Figure 1. Synthesis route of F127-Br.

Figure 2. Synthesis route of PNIPAM7-F127-PNIPAM7 (PN127).

and micelles with hydrophobic PNIPAM and PPO blocks as core and soluble PEO blocks as corona in dilute aqueous solutions at 20 and 40 °C, respectively, regardless of the relative lengths of PNIPAM, PPO, and PEO blocks. The size of “associate” structures of PNIPAM-P123-PNIPAM pentablock terpolymers at 20 °C increased with increasing length of PNIPAM block. Choi et al.12 synthesized PNIPAM-F68-PNIPAM for use as vehicle for controlled drug release. It was observed that critical micellar concentration increased with an increase in the length of the PNIPAM. Biocompatible amphiphilic pentablock copolymeric nanoparticles and vesicles of poly(lactide-coglycolide) and F68 for anticancer drug delivery were prepared by Byagari et al.13 Mallapragada et al.15 synthesized poly(amine methacrylate)-F127-poly(amine methacrylate) via controlled radical polymerization and used scattering and cryoTEM to examine temperature and pH-responsive self-assembly. They also studied micelles formed by pentablock copolymer poly[N,N-(diethylamino)ethylmethacrylate]-block-poly(ethyleneoxide)-block-poly(propyleneoxide)-block-poly(ethyleneoxide)-block-poly[N,N-(diethylamino)ethylmethacrylate]. F127 has been the Food and Drug Administration (FDA) approved and tried for delivery systems. It forms micelles of hydrodynamic diameter of ∼25 nm, which organize into thermoreversible gels at ambient temperatures. We attempted to carefully prepare a pentablock PNIPAM-F127-PNIPAM (hereafter referred to as PN127) with short PNIPAM block (DP = 7 as characterized by NMR). Pentablock copolymers can be used as nanocarrier for drug delivery system. We have carefully introduced seven NIPAM units at both PEO ends. We believe that if we introduce a longer PNIPAM group at both ends it may bend toward the PPO core, but with shorter PNIPAM group it may collapse on the surface and can be useful for controlled drug release. This copolymer’s monolayers were examined at air−water interface using Langmuir film balance and aggregation in water and salt solution by scattering techniques. The π-A isotherms at the air−water interface have been shown to provide useful information about the self-

assembly behavior of the copolymers and to predict the interactions with a variety of components in aqueous dispersion.16 Conformational changes and stability of the micelles in aqueous medium can be foreseen from the behavior of the monolayers, which make the analysis of the π-A isotherms a relevant tool for prediction of performance of polymeric micelles as drug nanocarriers. At low temperature the copolymer is expected to be more hydrophilic as compared with F127 due to hydrophilic PNIPAM end blocks, but above its LCST, PN127 is more hydrophobic than F127. The micelles of PN127 with collapsed PNIPAM may prove better substitute as nanocarrier for delivery system.

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EXPERIMENTAL SECTION Materials and Sample Preparation. Pluronic F127 (MW = 12 600, average number of PO unit = 65, average number of EO unit = 200),17 α-bromoisobutyryl bromide (98%), and tris[2-(dimethylamino)ethyl]amine (Me6TREN, 97%) from Aldrich (St. Louis, MO) were used without further purification. Triethylamine (99.0%) and CuBr (99.9%) from Wako Chemical (Osaka, Japan) were used without further purification. N-Isopropylacrylamide (NIPAM, 97%) from Aldrich was purified by recrystallization from a mixture of benzene and nhexane (3/7 v/v). Sodium chloride (NaCl) was from Merck (Darmstadt, Germany). Purified water (resistivity > 18.2 M Ohm·cm; MilliQ, Millipore Spain) obtained by reverse osmosis was used. All other reagents were of analytical grade. Synthesis of F127-Br. F127 (13.9 g, 1.10 mmol, MW = 12 600)17 was dissolved in dichloromethane (CHCl2, 50 mL). Triethylamine (0.33 g, 3.31 mmol) was added dropwise into the F127 solution under Ar atmospheres at 0 °C; then, 2bromoisobutyryl bromide (1.50 g, 6.52 mmol) was injected to the solution (Figure 1). The mixture was stirred at room temperature for 24 h. The reaction mixture was dialyzed against methanol and pure water. Br group-introduced F127 (F127-Br) was recovered by a freeze-drying technique (8.74 g, 63%). The 1 H NMR spectrum confirmed the successful preparation of F127-Br, as shown in Figure 3b. 7570


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Figure 3. 1H NMR spectra for (a) F127, (b) F127-Br, and (c) PN127 in CDCl3.

Synthesis of PNIPAM7-F127-PNIPAM7 (PN127). Tris[2(dimethylamino)ethyl]amine (Me6TREN, 9.25 μL, 0.0346 mmol) was dissolved in H2O (7.8 mL). The solution was stirred under an Ar atmospheres for 10 min. CuBr (6.71 mg, 0.0468 mmol) was then added. The stirring was continued for another 10 min. F127-Br (1.51 g, 0.118 mmol) and NIPAM (0.265 g, 2.34 mmol) in water (2.0 mL) were added to the Me6TREN and CuBr solution under an Ar atmosphere, and the mixture was then stirred at room temperature for 3 h (Figure 2). 1H NMR indicated that the conversion was 75%. The reaction solution was dialyzed against pure water for 3 days. PNIPAM7-F127-PNIPAM7 (PN127) was recovered by a freeze-drying technique (1.10 g, 62%). Methods. UV−Visible Spectroscopy. A Shimadzu model UV-2450 spectrophotometer including peltier system (Thermo Fisher) was used to record UV−visible absorption spectra to determine clouding behavior at different temperature. The peltier system was used to heat the cell holder of the spectrophotometer. The UV−visible absorption spectra of 1% PN127 and in 1 and 2 M NaCl solution at different temperatures were recorded. Micro-DSC. Microdifferential scanning calorimetry (microDSC) measurements were carried out by using a VP DSC (MicroCal) in Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China. The pentablock terpolymer was dissolved in deionized water to give an aqueous solution with concentration of 5%. The heating rate was 1.0 °C min−1 π-A isotherms. The experiments were carried out with a single barrier NIMA 611 surface balance (Coventry, U.K.), with total area 560 cm2, placed on an antivibration table. The surface pressure was measured with the accuracy of ±0.1 mN/m, using a Wilhelmy plate made from chromatography paper (Whatman Chr1, Brentford, U.K.) as a surface pressure sensor. Prior to experiments, the trough was thoroughly cleaned with ethanol and rinsed with water. Water and NaCl solutions 0.154 (0.9%, saline solution), 0.5, 1, and 2 M were used as subphase, and temperature values of 15, 20, and 30 ± 1 °C were set for the experiments. 80 μL of copolymer solution in chloroform (0.02%) was deposited at the air/water interface by means of a Hamilton (Reno, NV) syringe and allowed to stand for at least 10 min to ensure complete evaporation of the solvent. The solutions from which

monolayers were spread were made from others with a higher concentration, where chloroform was used as solvent along with 0.5% of amyl alcohol to improve extension. The monolayers were compressed at a constant rate of 15 cm2 min−1, and the surface pressure, π, was recorded as a function of the area of the monolayer and referred to the number of molecules. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were performed using a Malvern Instruments Zetasizer NaNO ZS equipped with a He-Ne laser (4 mW at 633 nm). Measurements were taken at a 173° scattering angle at different temperatures. F127 and PN127 were dissolved in pure water at a concentration of 5%. The water solutions were filtered using a membrane filter with 0.2 μm pores. Small-Angle Neutron Scattering. Small-angle neutron scattering (SANS) studies were performed at DHRUVA reactor, Trombay, India. The mean incident wavelength (λ) was 5.2 Å with wavelength resolution (Δλ/λ) ∼15% and measurements collected in the scattering vector (Q) range of 0.017 to 0.35 Å−1. The data were corrected for the background, the empty cell contributions, and the transmission. Correction due to the instrumental smearing was considered throughout the data analysis.18 The differential scattering cross section per unit volume (dΣ/dΩ) of monodisperse micelles in a SANS experiment is given by19 dΣ /dΩ = NP(Q )S(Q ) + B....

(1)

N is the number density of the micelles and B is a constant term that represents the incoherent background scattering mainly from the hydrogen present in the sample. P(Q) is the intraparticle structure factor characteristic of specific size and shape of the scatterers, and S(Q) is the interparticle structure factor that accounts for the interparticle interaction. The P(Q) is calculated using spherical hydrophobic core of micelle only. The scattering from hydrophilic shell is neglected because of very low contrast of shell compared with the core with respect to solvent.20 The aggregation number (Nagg) is calculated from the micellar core volume divided by the volume of hydrophobic PPO part of single block copolymer. The interparticle structure factor S(Q) for block copolymer micelles is captured by the analytical solution of the Ornstein−Zernike equation with the 7571


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The Journal of Physical Chemistry B Percus−Yevick approximation,21 employing hard sphere interaction Nagg =

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4πR c3 .... 3Vpo65

weight distribution (Mw/Mn) for the polymers are summarized in Table 1. The Mn(GPC) values are apparent values because Mn(GPC) is the relative molecular weight compared with standard polystyrene samples. The chemical structure of F127 and its derivatives is considerably different from that of standard polystyrene. Therefore, Mn(GPC) should be different from Mn(theo). Importantly, the Mn(NMR) value of PN127 was the same as the Mn(theo) value. This observation suggests that PN127 was well-controlled structure. Clouding Behavior. The CP is a physical property of block copolymer and can be tuned by regulating the copolymer composition and adding different additives. In general, in UV− visible spectrometer, the %T becomes zero as the CP of polymer is attained. Normally, CP was determined when percent transmission (%T) became zero in the temperature− transmission curve. For CP measurements, F127 and PN127 were dissolved in pure water and NaCl aqueous solutions, respectively. First, the CP values of F127 and PN127 were determined, and the transmission of copolymers aqueous solution remained 100% throughout the temperature range studied. The CP of Pluronic F127 (figure not shown), which is more than 100 °C, is in good agreement with the literature.22 In the case of PN127 (1%), the length of PNIPAM block is 7, so maybe it is too small to induce phase separation of copolymer below 100 °C (Figure 5a). Therefore, the length of PNIPAM block plays an important role in CP because it is soluble at lower temperature. Porjazoska et al.23 reported that copolymer of PL-PEO-PPO-PEO-PL series clouded at some temperature, which depended on the length of PL block. The CP of PN127 was also determined in the presence of NaCl solution. From Figure 5b,c, the CP decreased in the presence of NaCl. The addition of NaCl shifts the %T to lower value. From Figure 5b,c, the %T values for 1 and 2 M NaCl aqueous solution decreased with increasing temperature above 60 and 42 °C, respectively. The CP decreasing effect of NaCl can be quantitatively explained as due to the enhancement of water polarity by the less polarizable chloride ion.24 Micro-DSC. Figure 6 shows the micro-DSC data curve of 5% PN127 in aqueous solution during the heating and cooling process. This PN127 shows two thermal transition peaks with a strong endothermic peak centered at 19.8 and 34.5 °C. Alexandridis et. al22 reported that F127 showed CMT (critical micelle temperature) or LCST at 19.5 °C for 5% aqueous solution, which was attributed to PPO block. The LCST of PNIPAM homopolymer is around 32−34 °C in aqueous solution.25−27 Therefore, the first and second endothermic peaks could be attributed to the LCST of PPO block and PNIPAM block, respectively. A similar phenomena was observed by Du and coworkers. 28 They showed that PNIPAM110-F127-PNIPAM110 pentablock, a bimodal transition with a shoulder endothermic peak centered at 31 °C and a strong endothermic peak centered at 34 °C were observed,

(2)

RESULTS AND DISCUSSION Figures 3 and 4 show 1H NMR spectra and gel permeation chromatography (GPC), respectively. The GPC molecular

Figure 4. GPC charts for (a) F127, (b) F127-Br, and (c) PN127 using THF as the eluent.

weight distribution of F127 indicates a triblock PEO-b-PPO-bPEO and a low-molecular-weight PEO-b-PPO diblock copolymer fraction that is a result of the commercial synthesis process. GPC of F127 shows a small peak with retention time of ∼31.5 min in Figure 4. Mn and Mw/Mn of the small peak were 9530 and 1.02, respectively. Commercial sample of F127 contains a small amount of low-molecular-weight impurity, which is usually described as the diblock PEO-PPO component. We tried to remove the impurity by dialysis; however, the lowmolecular-weight compound could not be removed. Mw/Mn of PN127 was 1.15. When the polymerization was assumed to be ideally living process, the theoretical number-average molecular weight (Mn(theo)) and the theoretical values of degree of polymerization (DP(theo)) could be calculated from following equation. M n(theo) =

[M]0 conversion × × M m + MF127 [I]0 100

= DP(theo) × M m + MF127...

(3)

where [M]0 is the initial monomer concentration and [I]0 is the initial F127-Br concentration. Conversion is the percent conversion of the monomer, Mm is the molecular weight of the monomer, and MF127 is the molecular weight of F127. DP(NMR) was determined from the ratio of the integrated intensities of peaks b and h of Figure 3c. Mn, DP, and molecular

Table 1. Mn(theo), DP(theo), Mn(NMR), DP(NMR), Mn(GPC), and Mw/Mn for PN127 sample

Mn(theo)a

F127 F127-Br PN127

12600d 12797e 14400

DP(theo)a

7f

Mn(NMR)b

14400

DP(NMR)b

Mn(GPC)c

Mw/Mne

7

20700 23800 25100

1.20 1.15 1.15

a

Theoretical values estimated from eq 1. bDetermined by 1H NMR in CDCl3. cEstimated from GPC. dValue in catalog. eCalculated from the chemical structure. fDP(theo) of one PNIPAM block. 7572


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Figure 7. Both systems showed similar patterns, which are related to the consecutive conformational changes of the copolymers at the monolayer as the pressure increases. First, at an area ca. 6620 Å2/molecule (T = 20 °C) there is a flat (pancake) conformation of copolymer molecules spread on the interface, which offers low resistance to surface area contraction. When the surface area decreases, the pressure progressively increases up to a value at which a conformational change occurs. The copolymer then has to rearrange and adopts a mushroom conformation at an area ca. 4650 Å2/ molecule (T = 20 °C). In this region of the isotherm there is a slight increase in surface pressure while the surface area decreases. This pseudoplateau is interpreted as a rearrangement of the PPO coils into loops within the monolayer regime and the immersion of EO units in the aqueous subphase. The end of the pseudoplateau corresponded to the situation in which all EO groups become progressively immersed in the subphase, while the interface is exclusively occupied by the PPO blocks, in the case of F127. Further compression caused a rapid increase in the pressure that makes the copolymer molecules adopt a brush conformation at 2333 Å2/molecule (T = 20 °C) characterized by the stretching of the PPO due to space limitations and to increased lateral interactions.16 PN127 with seven NIPAM units grafted to each end of F127 showed π-A isotherms similar to those of F127, but the final slope was less steep and the extrapolated area was larger. This behavior can be related to the different polarities of the isopropyl (apolar) and amido (polar) groups of PNIPAM, which may have an effect on the overall conformation of the copolymer. At the beginning of the mushroom region, particularly at low temperatures, both PEO and PNIPAM chains protrude into the subphase, leaving PPO at the interface. The presence of PNIPAM as a second thermosensitive block at each end makes it rise out of the water as pressure rises, with each molecule occupying more area. For both F127 and PN127 copolymers, an increase in temperature from 15 to 30 °C at which PPO is less soluble in water led to an increase in the pressure recorded in the π-A isotherms. It is well known that temperature favors the entropydriven self-assembly process of F127 and PNIPAM polymers in separate, diminishing the micellar critical concentration of F127 and causing the CP of PNIPAM.29 Similarly, at the air−water interface the increase in temperature causes loss of hydration water from PO and NIPAM units, which, in turn, favors polymer−polymer interactions over polymer−subphase interactions. Strengthened hydrophobic interactions make the rearrangement of the copolymer and the compression at the interface more difficult during the contraction of the surface area. Overall, the increase in temperature led to monolayer expansion. Condensed phase appeared at greater value of surface pressure and mean molecular area for both F127 and PN127 (Figure 7). There was also an increase in the transition pressure, estimated from the minimum in the plots of compressibility modulus versus surface pressure.30 Values recorded for both copolymers at the three temperatures evaluated are summarized in Table 2. Effect of Ionic Strength. The effect of ionic strength on the behavior of monolayers was evaluated at a constant temperature of 30 °C and incorporating NaCl at various concentrations in the subphase, covering the physiological concentration of 0.9% (0.154 M). As shown in Figure 8, as ionic strength increased the monolayers progressively occupied

Figure 5. Percent transmittance (%T) at 600 nm for PN127 in (a) pure water and (b) 1 M and (c) 2 M NaCl as a function of temperature (Cp = 1%).

Figure 6. Temperature dependence of specific heat capacity for the 5% PN127 in aqueous solution.

which had been attributed to the LCSTs of the PPO block and PNIPAM block, respectively. Lu et al.11 showed the effect of different chain length of PNIPAM on LCST of PPO block in PNIPAMx-P123-PNIPAMx.They reported that as the chain length of PNIPAM block increases, the LCST of PPO shifted toward higher temperature. In our study we have only introduced small PNIPAM chain; therefore, it does not affect much the LCST of PPO block. π-A Isotherms. Effect of Temperature. The π-A isotherms of Pluronic F127 and the pentablock copolymer, PN127, at different temperatures, using water as subphase, are shown in 7573


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Figure 7. Isotherms of F127 and PN127 at the air−water interface recorded at various temperatures

Table 2. Surface Pressure Values for the Phase Transition in F127 and PN127 Monolayers at Different Temperatures

Table 3. Surface Pressure Values for the Phase Transition in F127 and PN127 Monolayers at Different Subphase NaCl Concentrations

πtrans (mN/m) temperature (°C) 15 20 30

F127 8.3 8.6 9.7

πtrans (mN/m)

PN127 9.0 9.7 10.6

greater surface area, the pressure values recorded at low area became greater, and πtrans shifted to higher values (Table 3). These findings correlate well with previously published data for other copolymers31 and can be related to the salting out effect that NaCl causes on the hydrophilic groups immersed in the subphase.32 The ions diminish the solubility of the copolymer in the aqueous medium, which leads to an increase in the area occupied at the air−water interface. The effect of increasing NaCl concentration in the subphase on the π-A isotherms of F127 and PN127 closely resembles the effect of raising the temperature, as both are related to dehydration of copolymer chains (PPO and both PPO and PNIPAM, respectively). Compressibility modulus (C s −1 ) of a monolayer is interpreted as the resistance of a monolayer to compression, as inverse to compressibility, and it is expressed as a function of the isotherm slope, in the same units as surface pressure

[NaCl] (M)

F127

PN127

0.154 0.5 1 2

9.7 10.5 11.4 14.3

10.5 11.0 11.9 14.9

Cs−1 = −A(dπ /dA)

where π is surface pressure and A is the mean molecular area. Changes in the compressibility modulus allow us to detect conformational rearrangements in the monolayer at the interface, and its plot against surface pressure displays local minima (when the monolayer is least resistant to compression) for phase transitions, as happens in the copolymers’ monolayer at different temperature and salt concentration. As seen in Figure 9, a clear phase transition at the pressure corresponding to the end of the pseudoplateau in the isotherm has been located for copolymer PN127 as well as for F127. That minimum is a result of a rearrangement caused by repulsion between PPO blocks in the top layer and PEO in the subphase.33 This leads to increased repulsions within the layer

Figure 8. Isotherms of F127 and PN127 at the air−water interface recorded at different salt concentrations. 7574


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Figure 9. Compressibility moduli (Cs−1) of F127 and PN127 monolayers on surface pressure (π).

Figure 10. Hydrodynamic diameter (Dh) distributions stack of PN127 in (a) water and (b) 1 M and (c) 2 M NaCl aqueous solutions at varying temperatures.

and the formation of loops. When aqueous subphase is replaced with NaCl solutions of increasing concentration, the minimum is shifted toward larger surface pressure, which means salt ions are dehydrating the PEO-PNIPAM chain. Therefore, less hydrated chains are less attracted toward the subphase, and hence it required more pressure for the phase transition. Dynamic Light Scattering. The DLS measurements on 5% solutions of F127 (figure not shown) and PN127 (Figure 10) in water and salt aqueous solution were made over a temperature range from 20 to 65 °C. These results are shown as stakes in the distribution profiles. At 20 °C both the copolymers showed a peak around ∼5 to 6 nm, indicating the presence of unimers along with a micelle peak around ∼20−25 nm plus some large clusters. This is quite expected for micelleforming Pluronic surfactants at temperatures close to CMT. Also, in the micro-DSC curve, it is observed that the LCST of PPO and PNIPAM is ∼19.8 and 34 °C, respectively, which implies that PPO is hydrophilic below the LCST temperature,

and above that they become hydrophobic and favor micellization. Therefore, small unimer peak is observed at lower temperature; however, at higher temperature the unimer peak disappears due to the predominance of the micelle peak. Even the larger clusters get solubilized in micelles, and a clearly distinct micelle peak can be seen from the DLS distribution plots. The scattering intensity increases significantly for both the copolymer solutions; the values were higher for PN127. The apparent hydrodynamic micelle radius varies slightly with the increase in temperature. This is due to two opposing effects. At higher temperature the copolymer becomes more hydrophobic so the aggregation number increases. At the same time increased temperature causes dehydration of hydrophilic shell, decreasing the shell thickness. In the presence of salt, PN127 shows a micellar peak around 22 nm, and the other peak is due to larger aggregates. It can be clearly seen that PN127 in salt behaves similar to that in water at various temperature ranges. 7575


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micelle structure at 28 °C, while in PN127, the scattering intensity shifted toward low q region, indicating the formation of larger spherical micelle structure. There is significant increase in the overall scattering with the increase in temperature. At 60 °C, the PPO blocks become completely dehydrated and formed spherical micelles core with corona of significantly hydrated PEO with hydrophobic PNIPAM block, resulting in attractive hydrophobic force between micelles. Therefore, at 60 °C, an insignificant amount PN127 remains as unimers, so growth in the size of micelles was likely due to increase in aggregation number. The PNIPAM end blocks are being hydrophilic below LCST (∼34 °C), and PEO blocks constitute heavily hydrated shell of the core−shell micelle; however, as the temperature increases PNIPAM becomes progressively hydrophobic, and above the LCST it collapses on the micelle surface. In the presence of NaCl, the scattering intensity of the peak shifted toward low q region as in temperature. This is an indication of further increase in aggregation number and agrees with light scattering data that core radius and aggregation number increase with temperature and salt concentration. One can also find the decrease in polydispersity as compared with F127 in PN127 that indicates that there was a significant number of micelles present in the PN127 solution. Both families of data, that is, F127 and PN127, showed the same trend at different temperature and salt concentration. Determan et al.14 have done SANS measurement of PDEAEM25-F127-PDEAEM25 at different pH and found that at low pH pentablock copolymer does not form micelles due to charge repulsion in the PDEAEM chain, while at higher pH it forms stable spherical micelles. Hadjiantoniou et al.,34 from SANS measurements, found that aggregation number and micelle radii are small for

With the addition of salt, PN127 shows a micellar peak at lower temperature and micelle size increases with temperature. Small-Angle Neutron Scattering. SANS measurement of 5% F127 and PN127 were done to investigate temperature- and salt-dependent evaluation of micellar structure (Table 4). Table 4. SANS Data for F127 and PN127 (5%) in Water and in Salt Solution at Different Temperature sample name

temperature (°C)

[NaCl] (M)

RC (nm)

polydispersity

Nagg

F127 F127 F127 F127 F127 PN127 PN127 PN127 PN127 PN127

28 60 28 60 28 28 60 28 60 28

0 0 1 1 2 0 0 1 1 2

4.4 5.3 5.4 5.6 5.5 6.1 6.4 6.3 6.6 6.9

0.41 0.31 0.34 0.27 0.32 0.33 0.24 0.30 0.16 0.26

57 100 105 117 111 151 180 167 192 220

Figure 11 shows the scattering data from 5% PN127 and F127 at different salt concentration and at two different temperatures. All data were fitted with spherical model for micellar core. The hydrophilicity of two blocks of PEO and PNIPAM in PN127 at lower temperature leads to lower scattering intensity. Hence scattering contribution from the unimers must be taken into account while fitting the data. The scattering from unimers has been modeled by the Gaussian chain. The Rg values of F127 and PN127 unimers were found to be 1.4 and 1.7 nm, respectively. In pure D2O, both copolymers show spherical

Figure 11. SANS pattern of 5% F127 (closed symbol) and PN127 (open symbol). 7576


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tri, tetra, and penta block copolymer, while those for di and hexa block copolymer are higher.

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CONCLUSIONS The present study thoroughly investigated the synthesis, adsorption, and aggregation behavior of pentablock copolymer. The copolymer with low polydispersity was synthesized by typical atom transfer radical polymerization (ATRP) method using NIPAM as monomer and modified F127 as the microinitiator. PN127 exhibits two LCSTs at 19.8 and 34.5 °C in aqueous solution, which can attribute to PPO blocks and PNIPAM blocks, respectively. PN127 displays an adsorption isotherm with four classical regions with the presence of pseudoplateau attributed to the pancake to brush transition as the PEO and PNIPAM chains penetrate into aqueous subphase. On the contrary, PN127 copolymer molecules tend to stay in the monolayer instead as dissolved in subphase in presence of salt, occupying greater area and generating more expanded monolayer as compared with F127. Aqueous solution of pentablock copolymer formed spherical micelles, as determined by DLS and SANS. The addition of PNIPAM block to PEO block resulted in increased in micellar size. The dehydration of PEO and PNIPAM blocks at higher temperature and salt concentration results in higher aggregation number as well as larger size of micelles.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 09879132125 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.P. thanks CSIR for providing senior research associateship. I.S.-M., M.C., and E.V.L. thank Susana Á lvarez-Perez for help with measurements on Langmuir monolayers.

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REFERENCES

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Surface and Aggregation Behavior of Pentablock Copolymer PNIPAM

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