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Exploring Aqueous Solution Dynamics of Amphiphilic Diblock Copolymer: Dielectric Relaxation and Time-Resolved Fluorescence Measurements EJAJ TARIF, Biswajit Saha, Kallol Mukherjee, Priyadarsi De, and Ranjit Biswas J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b00889 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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The Journal of Physical Chemistry

Exploring Aqueous Solution Dynamics of Amphiphilic Diblock Copolymer: Dielectric Relaxation and Time-Resolved Fluorescence Measurements Ejaj Tarif,1 Biswajit Saha,2 Kallol Mukherjee,1 Priyadarsi De2,* and Ranjit Biswas1,* 1

Chemical, Biological and Macromolecular Sciences (CBMS), S. N. Bose National Centre for Basic Sciences, JD Block, Sector III, Salt Lake, Kolkata-700106, India 2

Polymer Research Centre and Centre for Advanced Functional Materials, Department of

Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur741246, Nadia, West Bengal, India *Corresponding Authors. E-mails: [email protected] (RB), [email protected] (PD) Phone: +91 33 2335 5706 (RB); +91 967 462 9345 (PD); FAX: +91 33 2335 3477 (RB)

ABSTRACT: We explore in this work, after synthesizing and appropriately characterizing an amphiphilic diblock copolymer, the interaction of it with water molecules and the subsequent aqueous solution dynamics by employing time-resolved fluorescence measurements (TRF) and megahertz-gigahertz dielectric relaxation (DR) experiments. The synthesized amphiphilic diblock

copolymer

is

poly(2-(((tert-butoxycarbonyl)alanyl)oxy)ethyl

methacrylate)-b-

poly(polyethylene glycol monomethyl ether methacrylate) (P(Boc-L-Ala-HEMA)-b-PPEGMA)). Dynamic light scattering (DLS) measurements of aqueous solutions indicate formation of 14-20 nm particles, from a balance between the chain lengths of the hydrophobic (P(Boc-L-AlaHEMA)) and hydrophilic (PPEGMA) segments. Field emission scanning electron microscopy (FE-SEM), on the other hand, suggests a spherical shape for the dried micelles. The critical micelle concentration (CMC) of the P(Boc-L-Ala-HEMA)-b-PPEGMA block copolymer at different block lengths in aqueous media, determined via steady state fluorescence measurements, is very low (~4-8 mg/L), and the resultant micellar size has been found to be insensitive to the polymer concentration. Interfacial and bulk aqueous dynamics have been investigated by tracking the solution frictional resistance on rotational motion of dissolved hydrophobic and hydrophilic dipolar solute probes of comparable sizes. Time-resolved 1

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fluorescence anisotropy measurements reflect biphasic temporal profile for the frictional resistance. Interestingly, the hydrophobic probe, due to its preferential location at the micellar interface, experiences greater frictional resistance than the hydrophilic counter-part, although the latter reports stronger polymer concentration dependence of the frictional retardation than the former. DR measurements at the highest of the polymer concentrations considered suggest presence of aqueous dynamics slower than that for neat bulk water, although evidence for such “slow” dynamics at lower concentrations has not been detected in the present DR measurements. Keywords: Amphiphilic diblock copolymer, Synthesis, Aqueous solution dynamics, Time resolved fluorescence measurements, Dielectric relaxation

2


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INTRODUCTION Amphiphilic block copolymers, made of hydrophobic and hydrophilic segments, are well known to form ordered self-assembly such as cylinders, bicontinuous structures, micelles, vesicles, and other complex aggregations.1 Among these various nanostructures, micelles are the most studied morphologies in an aqueous medium, where micelle formation takes place upon appropriate hydrophobicity/hydrophilicity balance, wherein the impact of micelle hydrophobic block is compensated by the water soluble hydrophilic segment.2 Polymeric micelles have widely been considered as convenient nano-carriers for drug3 and gene delivery, 4 diagnostic imaging,5 etc. Admirable biocompatibility, lower critical micellar concentration (CMC) value, aqueous solution stability, high solubilization ability of a large number of hydrophobic drugs in their micellar core, etc. made polymeric micelle an excellent carrier in the biomedical field. 6,7 Compared to surfactant micelles, polymeric micelles are generally more stable, with a remarkably lowered CMC, and have a slower rate of dissociation, allowing retention of loaded drugs for a longer period of time and, eventually, achieving higher accumulation of a drug at the target site. 8 Unlike the conventional low-molecular weight surfactants and lipids, block copolymers have the advantage of modifying their shape and functionality depending on both their intrinsic properties (block-block interaction parameter) and extrinsic properties (molecular weight, block composition, solvent composition, concentration of the solution, etc.). Diverse biologically relevant self-organized aggregates such as micelles, 9,10 reverse micelles,11,12 vesicles,13,14 macromolecules and polymer aggregates,15,16 have frequently been used to study solvent dynamics using fluorescence spectroscopy. Among the different selfaggregate systems, micelles have been repeatedly used as model systems for confined reaction media. 17,18 Exploring dynamics in these media is critical because often micellar interface is heterogeneous, and this heterogeneity can considerably impact the course of a chemical reaction occurring at these interfaces. 19,20 In addition, micelle may be considered as a model bio-mimetic system for understanding the interaction of a biologically relevant species with the interface and subsequent transportation and release of it at a desired place. Thus, a thorough knowledge of microenvironment structure, dynamics and interaction with guest molecules is essential to control and/or suitably alter to meet the demands of daily care products, pharmaceutical and biomedical necessities. 3



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Neutron scattering, 21 nuclear magnetic resonance (NMR),22,23 fluorescence,24 static and dynamic light scattering 25 measurements have been used earlier to understand block copolymer micelles.26,27 Dynamics and interaction in solution phase of several different copolymers have been explored using a variety of ultrafast fluorescence techniques such as fluorescence resonance energy transfer (FRET),28 fluorescence correlation spectroscopy,29 fluorescence Stokes shift and anisotropy relaxations.30,31 Dielectric relaxation spectroscopy (DRS) probes the inherent medium dynamics,32,33 and has been utilized to explore polarization relaxations in a variety of systems that include pure solvents,34

ionic liquids, 35 deep eutectic solvents (DESs),36 organic

electrolytes, 37 polymer solution, 38 micelles,39 and reverse micelles. 40 However, a thorough and systematic investigation combining dielectric relaxation and pico-second resolved fluorescence spectroscopic techniques is still lacking for aqueous solutions of diblock copolymers. In order to provide such information of diblock copolymers possessing potential for applications, we have synthesized poly(2-(((tert-butoxycarbonyl)alanyl)oxy)ethyl methacrylate)-b-poly(polyethylene glycol monomethyl ether methacrylate) (P(Boc-L-Ala-HEMA)-b-PPEGMA)) diblock copolymer via reversible addition-fragmentation chain transfer (RAFT) polymerization and employ DR and TRF measurements for exploring interaction and dynamics of their aqueous solutions. We have used amino acid segment as a hydrophobic block because of their remarkable advantages, namely, good biocompatibility, non-toxicity and, more importantly, availability of side functional groups. The utility of these side functional groups is targeted for conjugation with molecules of biological relevance after the tert-butoxycarbonyl (Boc) deprotection followed by post-polymerization modification reactions.41 On the other hand, the oligo(ethylene oxide) side chains are uncharged, water-soluble, non-toxic, biocompatible, and thus advantageous towards smarter applications.42 EXPERIMENTAL SECTION Materials. Boc-L-alanine (Boc-L-Ala-OH, 99%, Sisco Research Laboratories Pvt. Ltd., India), 2-hydroxyethyl methacrylate (HEMA, 99%, Aldrich), 4-(dimethylamino)pyridine (DMAP, 99%, Aldrich), dicyclohexylcarbodiimide (DCC, 99%, Aldrich), anhydrous N,N'dimethylformamide (DMF, 99.9%, Aldrich), sodium bicarbonate (99%, Merck), coumarin 153 (C153, Aldrich), coumarin 343 (C343, Aldrich), pyrene (Aldrich) were used as received. Polyethylene glycol monomethyl ether methacrylate (PEGMA, 300 g/mol, Aldrich) was passed 4


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through

a

basic

alumina

column

prior

to

polymerization.

The

4-cyano-

(dodecylsulfanylthiocarbonyl)sulfanylpentanoicacid (CDP) was synthesized according to the procedure reported previously. 43 The initiator, 2,2′-azobis(2-methylpropionitrile) (AIBN, 98%, Aldrich) was purified by crystallization in methanol twice and stored in the refrigerator. For NMR study, CDCl3 (99.8% D) was purchased from Cambridge Isotope Laboratories, Inc., USA. Solvents such as hexanes (mixture of isomers), ethyl acetate (EA), dichloromethane, acetone, tetrahydrofuran (THF), etc. were purified by following standard procedures. The synthesis of 2(((tert-butoxycarbonyl)alanyl)oxy)ethyl methacrylate (Boc-L-Ala-HEMA) was conducted as reported elsewhere.44 RAFT Homopolymerization of Boc-L-Ala-HEMA. Boc-L-Ala-HEMA (1.5 g, 4.98 mmol), CDP (100.5 mg, 0.249 mmol), AIBN (4.08 mg, 0.0249 mmol), a magnetic bar and DMF (6.32 mL) were sealed in a 20 mL septum-capped glass vial at a feed ratio of [Boc-L-AlaHEMA]/[CDP]/[AIBN] = 20/1/0.1. The solution was purged with dry nitrogen for 12 min, and placed in a preheated block at 70 oC. After 3.5 h, the polymerization reaction was quenched by placing the vial in an ice-water bath, followed by opening the cap. The polymer, P(Boc-L-AlaHEMA) (1a), was isolated by precipitating the resulting solution in acetone/hexanes mixture followed by drying under vacuum at 40 oC for 8 h. We got 50% conversion, determined gravimetrically based on monomer feed. Synthesis of Amphiphilic Diblock Copolymers, P(Boc-L-Ala-HEMA)-b-PPEGMA (2a-2d). A typical block copolymer synthesis utilizing P(Boc-L-Ala-HEMA) as a macro-chain transfer agent (macro-CTA) is shown in Scheme 1. The PEGMA (332 mg, 1.1 mmol), P(Boc-LAla-HEMA) (320 mg, 55.3 µmol), DMF (1.37 mL) and AIBN (1.82 mg, 11.06 µmol, from stock solution) were sealed in a 20 mL septum-capped reaction vial at a feed ratio of [PEGMA]/[P(Boc-L-Ala-HEMA)]/[AIBN] = 20/1/0.2. The solution was purged with dry nitrogen for 12 min, and placed in a preheated reaction block at 70 oC. After a predetermined time interval, the polymerization reaction was quenched, purified and dried as mentioned above for the homopolymer synthesis. Similarly, another three block copolymers were synthesized by varying [PEGMA]/[P(Boc-L-Ala-HEMA)] ratios (40/60/90 : 1). Sample Preparation. For the preparation of micellar nanoaggregates in aqueous solution, de-ionized (DI) water was added drop-wise to the dried block copolymer and kept under stirring 5



Page 6 of 38

for about 10 min. Using this similar procedure, a series of micellar solutions of different concentrations (1, 5, 10 mg/mL) were prepared for all measurements at 25 oC. For optical measurements, the concentration of coumarin 153 (C153) and coumarin 343 (C343) were maintained to ~10−5 M in each sample. The structures of C153, C343, and pyrene are shown in Scheme 1.

Instrumentation and Methods Size Exclusion Chromatography (SEC). SEC was used to obtain molecular weights and molecular weight distributions (dispersity, Ð) of polymers in THF solvent at 30 oC at 1.0 mL/min flow rate. The SEC instrument contains a Waters 515 HPLC pump, a Waters 2414 refractive index (RI) detector, one PolarGel-M guard column (50 × 7.5 mm) and two PolarGel-M analytical columns (300 × 7.5 mm). The instrument was calibrated by using poly(methyl methacrylate) (PMMA) standards. Proton NMR Spectroscopy. All the 1H NMR spectra were acquired in a Bruker Avance III 500 spectrometer operating at 500 MHz. Dynamic Light Scattering (DLS). DLS measurements were conducted at 25 oC in a Malvern Nano Zetasizer instrument. The system was equipped with a He–Ne laser operating at a wavelength of 633 nm and a detection angle of 173o. Polymer solutions were filtered through a 0.45 µm syringe filter prior to measurement. Field Emission Scanning Electron Microscopy (FE-SEM). FE-SEM images were recorded in a Carl Zeiss Supra 55VP FE-SEM. An aliquot of sample solution (polymer concentration = 0.1 mg/mL in water) was drop casted on a silicon wafer, dried, coated with gold:palladium (20:80) alloy for imaging purpose. Density, Viscosity and Refractive Index Measurements. An automated temperature controlled density-cum-sound analyzer (Anton Paar, DSA 5000) was used for density measurements, and viscosity coefficients (η) were measured using a micro-viscometer (AMVn, Anton Paar).45,46 Refractive indices of the micellar solutions were measured using an automated temperature controlled refractometer (RUDOLPH, J357).47 6


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Steady State Measurements. Absorption spectra were recorded using UV-visible spectrophotometer (UV-2600, Shimadzu). Steady state emission spectra were obtained from fluorimeter (Fluorolog, Jobin-Yvon, Horiba). Before analysis, solvent blanks were subtracted from the spectra and converted properly to the frequency domain for further analysis and frequency determination.48,49 Time-Resolved Fluorescent Measurements. Time-correlated single photon counting (TCSPC) technique along with 409 nm (LED) excitation source was used for the time-resolved fluorescence measurements and the details of this setup are described elsewhere. 50,51 The full width at half-maximum (FWHM) of the instrument response function (IRF) obtained using 409 nm excitation sources and a scattering solution was ~80 ps. According to the established standard protocol52,53 for the anisotropy measurements, the time-resolved emission decays were collected at the peak wavelength of the steady state emission spectra. Emitted light at two different polarizations (with respect to the vertically polarized exciting light), known as vertically I para (t ) , and horizontally I perp (t ) polarized emissions, were collected. Then, the dynamic fluorescence anisotropy, r(t), was constructed as follows.54,55

r (t ) 

I para (t )  GI perp (t ) I para (t )  2GI perp (t )

.

(1)

The average rotational time,  rot , was obtained analytically from the constructed r (t ) via time integrating the bi-exponential fit parameters that described adequately the measured r (t ) for both the probes 



 r  dt [r (t ) r0 ] = 0



2

0

i 1

 dt   i exp(-t/ i ) = 11  22 with 1  2  1. The value of initial

anisotropy, r0 , was taken as 0.37654 for C153 and 0.3556 ( r0 value of C343 in glycerol, taken here as initial anisotropy) for C343. As concentration-dependent anisotropy measurements of different polymeric solutions may give different r0 value, we have fixed r0 for each probe molecule because we would like to compare the qualitative difference among the medium 7



Page 8 of 38

frictions resisting the solute rotating in different micellar solutions. G is the geometric factor and was obtained by tail matching of the intensity decays I para (t ) and I perp (t ) , and found to be 1.73  0.8 (for C153) and 1.54  0.7 ( for C343) respectively. Time-resolved fluorescence intensity decays of C153 in different polymeric solutions at magic angle (54.7 o) were also collected for average lifetime measurements. 57 Dielectric Relaxation Spectroscopy. The frequency dependent complex relative permittivity ε* (v ) is given as58

 * ( )   ' ( )  [i " ( ) 

ik ]. 2  P

(2)

Here,  represents the dc conductivity of the medium,  P the permittivity of free space. ε  and ε are respectively the permittivity and loss components of the complex frequency dependent dielectric function. All DR measurements were performed using a PNA-L Network Analyzer (N5230C) combined with a probe kit (85070E) operating in the frequency range 0.2 ≤GHz ≤50. Approximately 8 mL solution of each system was used, keeping all the external parameters fixed. Details regarding DR measurements can be found elsewhere. 59 Experimentally obtained ε* (v ) was then fitted with the following general equation.

  ( )    

n

 j

 1  (i2 j 1

j

)



1 j  j

,

58

(3)

where 0   j  1 and 0    1.  j represents the dispersion magnitude at the j -th relaxation step with the time constant,  j . Debye (D) relaxation corresponds to  j  0 ,  j  1 whereas  j  0 the Cole-Davidson (CD), and  j  1 the Cole-Cole (CC) model. Relaxation parameters were obtained via simultaneous fitting of ' ( ) and " ( ) by using a non-linear least squares method. Sufficient data points (~1000) were collected to get an accurate description from the fitting within the available frequency window. Also, data were collected both in the linear and logarithmic scales to cross-check the validity of the fit parameters and the accuracy of the fit descriptions. Quality

8


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of each of the fits were checked by examining the corresponding values of the ‘Goodness-of-fit’ parameter (  2 ) and residuals. The following expression60 defines 2 2 2 m    i    i   1    ,     2m   i 1   ( i)    ( i)     2

(4)

With m denotes the number of data triples ( , ,  ),  the number of adjustable parameters, i and ( i ) the residuals and standard deviations of the individual data points. For all

measurements presented here, 2D fits (2Debye processes) were employed to obtain the best simultaneous descriptions of the measured  ( ) and  () . Different combinations of Debye, Cole-Cole and Cole-Davidson processes were attempted but no better descriptions were found.

RESULTS AND DISCUSSION Synthesis and Characterization of Amphiphilic Diblock Copolymers. The amphiphilic diblock

copolymer,

P(Boc-L-Ala-HEMA)-b-PPEGMA

was

synthesized

via

RAFT

polymerization as shown in Scheme 1. We used RAFT technique to get molecular weight control during the homo- and block-copolymerization reactions.61 At first, the alanine-based methacrylate monomer (Boc-L-Ala-HEMA) was prepared using DCC/DMAP esterification reaction between HEMA and Boc-L-Ala-OH by following the procedure reported elsewhere (Scheme 1).44 Then, the homopolymerization of Boc-L-Ala-HEMA was carried out using CDP as a chain transfer agent and AIBN as an initiator at 70 oC in DMF at a feed ratio of [Boc-L-AlaHEMA]/[CDP]/[AIBN] = 20/1/0.1. The purity of the resulting homopolymer was confirmed by 1

H NMR spectroscopy (Figure 1A), where we did not observe any vinyl proton peaks of the Boc-

L-Ala-HEMA monomer. SEC analysis shows a unimodal molecular weight distribution with number-average molecular weight (Mn,SEC) of 3900 g/mol and Ð (Mw/Mn) of 1.11 (Figure S1). The number average degree of polymerization (DP) was found to be 18 from 1H NMR analysis by comparing the integration ratios of the chain end proton (-O-CH2-CH2-COOH) at 2.5 ppm with the methylene and methyne protons of the side-chain alanine moiety (peaks c, d and e) at 4.1-4.5 ppm (Figure 1A). This result assists us to measure the number-average molecular weight from NMR (Mn,NMR) as 5820 g/mol. 9



Page 10 of 38

Next, P(Boc-L-Ala-HEMA) was employed as a macro-CTA to polymerize PEGMA at 70 o

C in DMF to afford a series of amphiphilic diblock copolymers (Scheme 1). The macro-CTA to

initiator ratio = 1:0.2 was kept constant during all the polymerization reactions. After the polymerization reaction, the unreacted monomers were removed by precipitating the copolymers in acetone/hexanes mixture. Finally, the purified block copolymers were characterized by 1H NMR spectroscopy and SEC analysis. A typical 1H NMR spectrum of 2a is shown in Figure 1B, where the appearance of resonance signals from both the block segments are assigned on the spectrum. Also, the absence of any vinyl peak in the region of 5.5-6.5 ppm confirms the purity of the block copolymer. Furthermore, 1H NMR analysis was applied to determine the DP of the PPEGMA segment. The DP was calculated on the basis of the integral peak ratios between the chain end proton (-O-CH2-CH2-COOH) at 2.5 ppm and the methoxy proton (peak m) of PEGMA unit at 3.37 ppm. Subsequently, the calculated DP of PEGMA in combination with the molecular weight of P(Boc-L-Ala-HEMA) macro-CTA assist us to determine the Mn,NMR values of block copolymers (Table S1). The theoretical molecular weights (Mn,theo) were calculated based on monomer conversion by using the formula; Mn,theo = (([PEGMA]/[macro-CTA] × molecular weight (MW) of PEGMA × conversion) + MW of macro-CTA). The Mn,theo values are summarized in Table S1 matches nicely with the corresponding Mn,NMR values. The SEC traces of the diblock copolymers revealed a monomodal elution peak and a clear shift towards the higher molecular weight region compared to the P(Boc-L-Ala-HEMA) macro-CTA (Figure S1). Moreover, we did not observe any noticeable bimolecular termination products or unreacted macro-CTA, thus supports successful block copolymerization As expected SEC RI traces shifted to lower elution volume with the increase of [PEGMA]/[macro-CTA] (20/40/60/90:1) ratio (Figure S1). The Mn,SEC and Ð values of the block copolymers are included in Table S1, where the Mn,SEC values of the diblock copolymers are somewhat lower compared to the respective Mn,theo values. This could be attributed to the difference in hydrodynamic volume of as-synthesized diblock copolymers with the PMMA standards which we have used to construct the calibration curve.

10


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Self-Assembly Behaviour of the Diblock Copolymers. The synthesized diblock copolymers, 2a-2d, contain hydrophilic (PPEGMA) and hydrophobic (P(Boc-L-Ala-HEMA)) segments, thus expected to undergo self-assembly under proper conditions. All the diblock copolymers are completely soluble in water without any external assistance, because of the significant introduction of the hydrophilic PPEGMA entity into all the copolymer chains. The critical aggregation concentration (CAC) of the copolymers were determined by virtue of fluorescence spectroscopy (Figure S2) using pyrene as a luminescent probe due to its high sensitivity to the microenvironment polarity change. 62,63 As illustrated in Figure S3, the CAC of 2a-2d were measured from the cross-section of two linear fitting curves of I388/I368 vs. log(polymer concentration) plot. Notice that increase in hydrophilic length leads to a higher CAC value (Table S2). This is expected because increased hydrophilicity induces better dissolution of these amphiphilic polymer molecules by the aqueous environment. For these polymeric micelles, lower CAC value gives an advantage for its application as a carrier as lower CAC may help to resist dissociation on dilution inside the body fluid.64 In the next step, the size of the self-assembled particles in an aqueous medium from 2a-2d was investigated by DLS measurement. The number-average hydrodynamic diameters, Dh, were in the range of 14±0.8 nm (polydispersity index, PDI = 0.459) to 20±1.0 nm (PDI = 0.333) in an aqueous medium at a polymer concentration of 1.0 mg/mL (Figure S4) and concentrationdependent Dh values are tabulated in Table S2. These Dh values are above the threshold value (~5.5 nm) of excretion limit and avoid renal excretion.65 Furthermore, these values are much lower than the 100 nm and help to bypass RES (reticuloendothelial systems) uptake.66 Nevertheless, the morphologies of the self-assembled particles were explored by FE-SEM observations (Figure 2). FE-SEM images of 2a-2d exhibited uniform spherical micelles with a diameter in the range of 40-60 nm. The aforesaid noteworthy dissimilarity in the sizes obtained from DLS and FE-SEM study may be attributed to the different technique of size measurement.67 Though in FE-SEM high vacuum leads to the water loss and obtained microstructure is actually for the dried micelle. Nevertheless, these studies confirmed micelle formation from 2a-2d in water.

11



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Steady State Absorption and Emission. Figure 3 represents the absorption and emission spectra of coumarin 153 (C153) in aqueous micellar solutions of diblock copolymer (1 mg/mL). Average spectral peak frequencies at different polymer concentrations (1, 5 and 10 mg/mL) are summarized in Table 1. For comparison, steady-state absorption and emission spectrum of C153 in pure water are also shown in the same figure. Clearly, the absorption spectra of C153 in aqueous polymer solutions show a much weaker blue-shift (compared to that in neat water) than that for the corresponding fluorescence spectra. Blue shift of emission spectra in these polymeric micellar solutions (relative to that in water) suggests local environments probed by C153 are less polar than water. This suggests that C153, due to its hydrophobic nature, locates itself at the micelle-water interface. This blue shift in emission spectra relative to water has already been observed for bile salt aggregates,68 and micellar mixtures.69 Note that the extent of spectral blueshift does not depend upon the number of hydrophilic chain (as for 2a, 2b, 2c and 2d) in these aqueous polymeric micellar solutions. This observation provides a support in favour of C153 being located at the micellar interface. A sharp increase of emission intensity over that in bulk water (controlled experiments with same C153 concentration), shown in Figure S5, further supports this view of C153 location in these polymeric micellar solutions. Excitation wavelength dependence of fluorescence emission of C153 in these micellar solutions (shown in Figure S6) suggests that, these solutions, within the lifetime of C153, are spatially homogeneous in nature. Here, spatial homogeneity refers to the ‘uniformity’ in microenvironments that surround the probe molecules in different micellar aggregates present in the solution. If microenvironments around probe molecules in different aggregates in solution are similar or the interconversion among dissimilar environments is much faster than the average excited state lifetime of the probe, then the fluorescence emission occurs from the excited probe molecules surrounded by a completely relaxed, fluctuation-averaged ‘single’ environment. This leads to insensitivity of the fluorescence emission energy to the choice of probe excitation wavelength. This is reflected in our emission spectra (Figure S6) collected after varying the excitation wavelength. Note the emission peak frequencies corresponding to different excitation wavelengths are nearly similar (within the uncertainty of  250 cm-1). An excitation wavelength dependence of fluorescence emission is then interpreted in terms of differing solvation environments (spatial heterogeneity) surrounding the dissolved probe molecules. 12


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Solute Rotation and Probe Location. Dynamic fluorescence anisotropy measurements provide an estimate of the restriction experienced by a dissolved solute while undergoing rotation in its own environment.70 Representative time-resolved fluorescence anisotropy decay ( r (t ) ) for C153 in one of these micellar solutions and the corresponding bi-exponential fit is shown in Figure 4. Figure S7 presents the corresponding intensity decays at the parallel and perpendicular polarizations for this solution. r (t ) decays at other polymeric micellar solutions are provided in Figure S8. Bi-exponential fit parameters for the measured r (t ) decays are summarized in Table 2. Note these bi-exponential anisotropy decays are characterized by a dominant (~60%) fast relaxation (~60 picoseconds) component and a relatively slower nanosecond component. This behaviour of relaxation represents the bimodal nature of frictional resistance of the local environment to the rotational motion of the solute. It is also clear from Table 2 that increase of the number of hydrophilic chain in polymer does not have significant impact on the average rotational time (  r ) of the probe (C153). Such an observation indicates that C153 resides near to the hydrophobic surface, supporting the conclusion from steady state spectroscopic results. Furthermore, average rotational time (~ 1 ns) of C153 in this polymeric micelle is approximately 10-15 times greater than that (~50 ps)53 for C153 in neat water, although the bulk viscosity of the aqueous polymeric solution (shown in Table S3) is comparable to that of neat water. 53 This supports the view that C153 in these polymeric solutions resides at the water-micelle interface. Polymer concentration dependent rotational anisotropy decays, shown in Figure 5 and biexponential fit parameters in Table 2, indicate that increase of polymer concentration slows down the probe rotation. Interestingly, the nanosecond timescale becomes longer with polymer concentration whereas the sub-100 ps timescale shows relatively much weaker concentration dependence. This is due to the increase in the number of micellar aggregates (“crowding”) with polymer concentration which is consistent with the DLS results that indicate near-insensitivity of the micellar size to the polymer concentration but increase in scattering intensity (Table S2). For further confirmation of probe location, dynamic anisotropy measurements using a hydrophilic probe coumarin 343 have been carried out that produced average rotation times in the ~300-800 ps range (shown in Table S4). This much faster average rotation time for a probe (C343) comparable to the size of C153 indicates C343 preferentially locates in the more labile water-like environments.54,71 However, these C343 environments in these aqueous solutions are neither 13



Page 14 of 38

bulk water-like nor like those for C153 which penetrates deeper due to hydrophobicity, but rather away from the deep palisade layer and toward the outer periphery of the micellar interface. Formation of more micellar structures at higher concentrations (“crowding”) is likely to reduce the mobility of

the interfacial water molecules, inhibiting further the solute rotation.

Concentration-dependent rotational anisotropy decays of C343 are shown in Figure 6. Data summarized in Table S4 reflects that average rotational time for C343 slows down with polymer concentration, which corroborates well with the observation of lengthening of C153 rotation times in these solutions. Due to much faster medium relaxation compared to the time resolution available, we were unable to detect the Stokes shift dynamics for the solutions under study. Average fluorescence lifetime decays for C153 in these solutions have been collected. Excited state average lifetimes,  lif  obtained from time-integrating the tri-exponential fits to the lifetime decays, are

summarized in Table 2. Individual tri-exponential fit parameters are provided in Table S5 (supporting information). Note in these tables that not only the average lifetimes are similar in all these polymeric micellar solutions but also the individual amplitudes and time constants remain nearly insensitive to the polymer concentration. This suggests micro-environments surrounding the probe are similar in all these aqueous solutions. This corroborates well with the view that C153 resides at the interface, and polymer concentration cannot alter this preferential location for C153. Dielectric Relaxation. Figure 7 represents the real (  ' ) and imaginary (  ' ' ) components of the complex dielectric spectra of polymeric micellar solutions (10 mg/mL) along with the 2-Debye fits. Fit parameters are shown in Table 3. Tabulated data suggest two DR time scales, and they are in ~40 ps and ~10 ps ranges. This may represent two different relaxation mechanism (involving the same or different species) in these micellar media. Note this ~40 ps component, although changes slightly with the identity of the polymer, is too small to be considered seriously. However, a comparison between the residuals obtained from single and two Debye fits (Figure S9) suggests that the presence of this slow component might be real. If we allow for this then the relatively faster ~10 ps timescale may be attributed to the DR of bulk-like water as separate DR measurements of neat water in this frequency window provide a single Debye 14


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relaxation with a DR time ~9 ps (see Table 2). 72 The slower ~40 ps timescale then may be considered to originate from the relaxation of those water molecules which are residing at the micellar interface. Note such slower relaxations have been observed earlier in aqueous solutions containing biologically relevant molecules such as lipids, proteins, nucleic acids etc. and confined water systems (micelle, reverse micelle etc).73 The small, less-than-a-percent, amplitude of the slow relaxation observed here may indicate that the population of water molecules residing in the interfacial regions is extremely small. Such a negligible population of interfacial, “slow” water molecules might be the reason for us not being able to detect the Stokes shift dynamics in these polymeric micellar systems. This may be one of the significant differences between the micelles created by these diblock copolymers, and those by the conventional more-studied surfactants.20,33 Note also that the measured reorientational dynamics of C153 in these aqueous micellar solutions is faster than that observed with the same probe in non-ionic aqueous micellar solutions of Triton X 100 (TX100) and n-octyl-β-D-thioglucoside (OTG) of comparable viscosity.69 These differences in solute-centred dynamics (solute rotation and solvation) between the classical surfactant solutions and the present diblock copolymeric systems warrant further study. CONCLUSIONS In conclusion, megahertz-gigehertz dielectric relaxation and pico-second resolved fluorescence measurements were used to explore and understand the solution dynamics in aqueous milieu containing P(Boc-L-Ala-HEMA)-b-PPEGMA diblock copolymers with tunable segment lengths. Two coumarin probes, C153 (hydrophobic) and C343 (hydrophilic), have been employed independently to explore the different environments in these solutions and the corresponding magnitudes of the frictional resistance exerted by the different local environments.

An

introduction of appropriate amphiphilicity into copolymer permits to investigate their selfassembly behaviour in water. The synthesized diblock copolymers formed well-defined spherical micelles of nanoscopic size. The hydrophobic probe, due to its preferential location at the micellar interface, experiences greater frictional resistance than the hydrophilic counter-part, although the latter reports stronger polymer concentration dependence of the frictional retardation than the former. Steady state and time-resolved fluorescence spectroscopic results suggest that the hydrophobic probe resides at the water-micelle interface whereas the hydrophilic 15



Page 16 of 38

probe prefers to locate in the water-like environment. In addition, DRS results suggest possible presence of “slowed down” water molecules at the interfacial region although the population of such “slow” water molecules in this region might be very small. Such thin hydration layers around the aggregates possessing relatively smaller hydrodynamic radii may have specific use as a carrier through tortuous path of nanometer dimension.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge in the ACS publications website at DOI:-----. SEC RI traces of polymers, details of block copolymerization, fluorescence spectra of encapsulated pyrene into micelles, determination of CMC value, DLS curves of copolymers 2a2d and Particle size distribution. Steady state emission spectra of C153, excitation wavelength dependence of C153 emission spectra, fluorescence intensity decay profiles for constructing anisotropy decays for C153 in aqueous polymer solutions and the corresponding rotational anisotropy decay profile (representative), densities, viscosities, and refractive indices for the solutions studied; average rotational time and the associated decay components for C343, and average lifetime and emission intensity decay (magic angle) components of C153 as a function of concentration; comparison (representative) between single and double Debye fits of real and imaginary components of DR spectra.

16


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AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected]

*

E-mail: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS B.S. acknowledges Council of Scientific and Industrial Research (CSIR), Government of India, for his Senior Research fellowship. One of us (RB) thanks Professor A. Barman, S. N. Bose National Centre for Basic Sciences, for helping us to record the DR spectra presented here.

17



Page 18 of 38

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Scheme 1. (A) Synthetic route employed for the synthesis of P(Boc-L-Ala-HEMA) and its block copolymerization with PEGMA by RAFT polymerization. (B) Structures of the external probe molecules used in the study.

26


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Table 1. Absorption and Emission peak frequencies of C153 in different polymeric (2a, 2b, 2c, 2d) micelle solutions at three different concentrations at ~25 oC. Absorptionm

Emissionm

Peak Frequency (103 cm-1)

Peak frequency (103 cm-1)

Polymer

m

1 mg/mL

5 mg/mL

10 mg/mL

1 mg/mL

5 mg/mL

10 mg/mL

2a

23.57

23.59

23.59

19.69

19.87

20.00

2b

23.63

23.67

23.64

19.64

19.79

19.87

2c

23.68

23.79

23.68

19.64

19.72

19.75

2d

23.72

23.67

23.73

19.45

19.70

19.74

These data are reproduced within the uncertainty of

 250 cm-1 (based on 2-3 independent measurements).

27



Page 28 of 38

Table 2. Parameters from bi-exponential fits to concentration-dependent anisotropy decays measured using C153 in different polymeric (2a, 2b, 2c, 2d) micellar solutions at ~25 oC. Polymer

1 (%)

 1 (ns)

 2 (%)

 2 (ns)

 r ( ns) n

 lif (ns) o

1

60

0.070

40

2.04

0.86

3.34

5

58

0.055

42

2.36

1.02

3.20

10

70

0.052

30

4.38

1.35

3.42

1

57

0.079

43

2.41

1.08

3.64

5

61

0.065

39

3.29

1.32

3.70

10

64

0.063

36

4.09

1.51

3.81

1

59

0.069

41

2.24

0.96

3.80

5

65

0.062

35

3.52

1.27

3.93

10

66

0.059

34

3.80

1.33

3.85

1

61

0.096

39

2.31

0.96

3.96

5

64

0.069

36

2.78

1.04

3.84

10

75

0.051

25

4.40

1.14

3.68

Conc. (mg/mL)

2a

2b

2c

2d

n

Fit parameters have been obtained after fixing the r0 values at 0.376 (C153) and 0.35 (C343). Individual time

constants are better than ±8% of the reported values. oThese data can be reproduced within the ±5% uncertainty.

28


Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Parameters obtained from simultaneous 2D fits to real (  ) and imaginary (  ) components of the measured DR spectra for aqueous polymer solutions at 10mg/mL polymer concentration and ~25 oC. Polymer

s

 1

 1 (ps)

u

 2

 2 (ps)



nD

(10 mg/mL)

 fit

2

(S/m)

2a

79.4

0.20

47

73.7

9.6

5.5

1.264

0.01

0.013

2b

78.5

0.20

34

73.3

8.9

5.0

1.264

0.01

0.009

2c

78.6

0.40

36

73.1

8.9

5.1

1.265

0.01

0.005

2d

80.0

0.44

37

73.9

9.9

5.7

1.264

0.01

0.005

Water (~22 oC)

80.1

-

-

75.4

9.3

4.7

1.263

-

0.062

 i (i=1-2)

u

are better within ±5% of the reported values (based on 2 to 3 independent measurements).

29



Page 30 of 38

Figure Captions Figure 1. 1H NMR spectra of (A) P(Boc-L-Ala-HEMA) and (B) P(Boc-L-Ala-HEMA)-bPPEGMA (2a) in CDCl3 Figure 2. FE-SEM images of 2a and 2c. Samples were prepared using 0.1 mg/mL polymer concentration in water. Figure 3. Absorption and emission spectra of C153 in aqueous micellar solutions of diblockcopolymers (2a, 2b, 2c, 2d) with 1 mg/mL polymer concentration. Spectra of different polymers are color-coded. Black dashed lines represent absorption and emission spectrum of C153 in water. Figure 4. Representative time-resolved fluorescence anisotropy ( r (t ) ) decay for C153 in aqueous polymer solution (2b). Black line going through the data denotes bi-exponential fit. Figure 5. Polymer concentration dependence of rotational anisotropy ( r (t ) ) decay for C153 in aqueous polymer solution (2b). Decays at different concentrations (mg/mL) are color-coded. Solid line represents fit. Figure 6. Time-resolved fluorescence anisotropy ( r (t ) ) decays for C343 in aqueous polymer solution (2b) at different concentrations (1, 5 and 10 mg/mL). Decays at different concentrations are color-coded. Lines going through the data represent fits. Figure 7. Real (  ) and imaginary (  ) components of the measured DR spectra for aqueous micellar solutions of different polymers (2a, 2b, 2c, 2d) at 10 mg/ml concentration. Measurements were done in the frequency regime, 0.2   / GHz  50 and at ~ 25 oC. Solid lines through these data represent simultaneous fits using 2D relaxation model. Note the collected DR spectrum for neat water (pink) is nearly indistinguishable from those for polymer solutions.

30


Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1/Tarif et al

31



Page 32 of 38


32


Page 33 of 38

1 .0

N o r m a liz e d I n t e n s it y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A b s.

Em.

C 153

C 153

0 .8

0 .6

0 .4

0 .2

20

22

2 a 1 m g /m L

2 a 1 m g /m L

2 b 1 m g /m L

2 b 1 m g /m L

2 c 1 m g /m L

2 c 1 m g /m L

2 d 1 m g /m L

2 d 1 m g /m L

W a te r

W a te r

24

26

28 16

17 3

18

19

20

21

22

-1

F r e q u e n c y (1 0 c m )


33



0 .4 C 153 2 b 1 m g /m L

0 .3

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

0 .2

0 .1

0 .0

0

1

2

3

4

5

6

T im e ( n s )


34


Page 35 of 38

0 .4 C 153 2 b 1 0 m g /m L 2 b 5 m g /m L

0 .3

2 b 1 m g /m L

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 .2

0 .1

0 .0

0

1

2

3

4

5

6

T im e ( n s )


35



0 .4 C 343 2 b 1 0 m g /m L 2 b 5 m g /m L 2 b 1 m g /m L

0 .3

0 .2

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

0 .1

0 .0

- 0 .1

0

1

2

3

4

5

6

T im e ( n s )


36


Page 37 of 38

80 ' 60

' and "

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

W a te r 2 a 1 0 m g /m L 2 b 1 0 m g /m L 2 c 1 0 m g /m L 2 d 1 0 m g /m L

"

20

0 1

10

 /G H z


37



Page 38 of 38

TOC Graphic

38


Dielectric Relaxation and Time-Resolved ... - ACS Publications

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