Thermoregulated Formation and Disintegration of Cationic Block

Article pubs.acs.org/JPCB

Thermoregulated Formation and Disintegration of Cationic Block Copolymer Vesicles: Fluorescence Resonance Energy Transfer Study Chiranjit Maiti, Debabrata Dey, Sarthak Mandal, and Dibakar Dhara* Department of Chemistry, Indian Institute of Technology Kharagpur, West Bengal 721302, India S Supporting Information *

ABSTRACT: Formation and disintegration of self-assembled nanostructures in response to external stimuli are important phenomena that have been widely explored for a variety of biomedical applications. In this contribution, we report the thermally triggered assembly of block copolymer molecules in aqueous solution to form vesicles (polymersomes) and their disassembly on reduction of temperature. A new thermoresponsive diblock copolymer of poly(N-isopropylacrylamide) poly((3-methacrylamidopropyl)trimethylammonium chloride) (PNIPA-b-PMAPTAC) was synthesized by reversible addition−fragmentation chain transfer technique. The solution properties and self-assembling behavior of the block copolymer molecules were studied by turbidimetry, temperature-dependent proton nuclear magnetic resonance, fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy. Fluorescence resonance energy transfer studies between coumarin-153 (C-153, donor) and rhodamine 6G (R6G, acceptor) have been performed by steady-state and picosecond-resolved fluorescence spectroscopy to probe the structural and dynamic heterogeneity of the vesicles. The occurrence of efficient energy transfer was evident from the shortening of donor lifetime in the presence of the acceptor. The capability of the vesicles to encapsulate both hydrophobic and hydrophilic molecules and release them in response to decrease in temperature makes them potentially useful as drug delivery vehicles.

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of drug molecules.8,21,22 In general, vesicles are formed with the involvement of various organic solvents such as tetrahydrofuran, N,N-dimethylformamide, and 1,4 dioxane,15,23,24 which, however, are not preferred because of their toxicity and cost ineffectiveness due to the requirement of elaborate purification and dialysis. Furthermore, dialysis rate and solvent dependence on self-assembly is difficult to control. In this regard, watersoluble block copolymers that spontaneously form vesicles are highly desirable, as vesicles may be prepared without involving organic solvents. Various ultrafast fluorescence studies such as photoinduced electron transfer, solvation and rotational dynamics, fluorescence resonance energy transfer (FRET),25 and fluorescence correlation spectroscopy26 have been explored to understand the structural and conformational dynamics of different nanostructures of block copolymers.27−29 Among these, FRET is used as a powerful tool to determine the distances between donor and acceptor, especially when they are covalently labeled at the two specific sites of a macromolecule.30 However, it has also been used to probe the structural dynamics of many nanostructured assemblies such as, micelles, reverse micelles, microemulsions, and vesicles that are

INTRODUCTION Stimuli-responsive polymers have been attracting tremendous research interest due to their fascinating phase-transition properties in the presence of external stimuli such as temperature, pH, light, electric potential, and magnetic field.1−5 In particular, the area of thermoresponsive polymers is a steadily growing field in polymer research, particularly the studies around the lower critical solution temperature (LCST) exhibited by the thermoresponsive polymers.6−10 A block copolymer containing thermosensitive block is rendered amphiphilic below or above the LCST depending on the nature of the other block11 and subsequently exhibits spontaneous self-organization forming interesting micellar or vesicular nanostructures in aqueous solution.12−14 The vesicles or polymersomes formed by amphiphilic block copolymers are usually spherical, with a hydrophilic core, hydrophobic shell, and an external hydrophilic corona15−17 that enable them to encapsulate both hydrophilic and hydrophobic drug molecules. Cationic vesicles are important because of the possibility of DNA uptake in their interiors, thus favoring nonviral gene-delivery in biological systems.18−20 Polymeric vesicles, which respond to external stimuli such as a change in temperature or pH, represent one of the most attractive candidates for application in drug delivery systems. Disassembly of vesicles in response to a stimuli may result in the release of the payload from a vesicle, thereby triggering targeted delivery © 2014 American Chemical Society

Received: December 16, 2013 Revised: February 3, 2014 Published: February 3, 2014 2274

dx.doi.org/10.1021/jp412273h | J. Phys. Chem. B 2014, 118, 2274−2283

The Journal of Physical Chemistry B

Article

Scheme 1. Schematic Representation of Synthesis of Diblock Copolymers (PNIPA-b-PMAPTAC) Using NIPA and MAPTAC by RAFT Processa

a

Chemical structures of FRET donor (C-153), acceptor (R6G), and Nile Red are also shown.

biologically relevant.31−34 Because energy transfer phenomenon is strongly dependent on the distance between the molecular centers of donor and acceptor, successful application of energy transfer processes requires precise control over their relative positions. Self-assembly of block copolymer is very useful for the precise positioning of the fluorophores that enables the FRET process to occur. In these assemblies, there exists a distribution of donor−acceptor (D−A) distances instead of a single D−A distance. Despite these complexities, the calculations of multiple D−A distances are potentially important in studying self-assembled systems with fluorophores. It is possible to get some insight into the structural and dynamic heterogeneity of such self- assembled systems by monitoring FRET parameters. In the present work, we report the formation of vesicles (polymersomes) from a new thermoresponsive diblock copolymer, poly(N-isopropylacrylamide)-block-poly((3methacrylamidopropyl)trimethylammonium chloride) (PNIPA-b-PMAPTAC), synthesized by reversible addition− fragmentation chain transfer (RAFT) technique. We showed that these vesicles can be used to encapsulate both hydrophobic and hydrophilic molecules that can be released simply by reducing the temperature of the aqueous solution. We have also studied FRET between C-153 (donor) and R6G (acceptor) in these thermoresponsive cationic diblock copolymer vesicles to construct an idea about the rate and efficiency of energy transfer inside these cationic vesicles as well as to get some insight about the microenvironment of the donor and acceptor molecules embedded in the self-assembled nanostructures that have biological significance.

NMR spectra are provided in the Supporting Information (Figures S1 and S2). Milli-Q water was used for sample preparation. C-153, R6G, and Nile Red were dissolved in methanol for the preparation of stock solutions. Aqueous solutions of the probe were prepared by taking appropriate aliquots of the probe from the stock and evaporating methanol using a stream of nitrogen gas. Aqueous polymer solution of desired concentration was added to achieve the required final probe concentration. The structures of C-153, R6G, and Nile Red are shown in Scheme 1. Synthesis of Cationic Diblock Copolymer (PNIPA-bPMAPTAC, Scheme 1). At the first step, poly(N-isopropylacrylamide) macro chain transfer agent (CTA) was synthesized at 70 °C using ACVA and DDMAT as the initiator and primary CTA, respectively. The polymerization was carried out in 1,4dioxan for 16 h under a nitrogen atmosphere with a monomer (NIPA) to CTA ratio ([M]0/[CTA]0) of 120:1 and CTA to initiator ratio ([CTA]0/[I]0) of 3:1. The polymerization reaction was quenched by freezing the solution in liquid nitrogen. Then, the reaction mixture was diluted with minimum volume of THF, and the product was obtained by precipitating the solution into an excess amount of ice-cold diethyl ether. The precipitation process was repeated thrice; then, the solid product was filtered. The final product was collected after vacuum drying overnight. In the next step, the block copolymer with desired number of MAPTAC repeat units was synthesized by polymerizing MAPTAC in the presence of poly(Nisopropylacrylamide) macro-CTA (1:100 mol ratio of CTA/ MAPTAC) in DMF/H2O (3:1 v/v) mixture for 24 h at 70 °C under a nitrogen atmosphere. The reaction mixture was again quenched in liquid nitrogen and dialyzed against distilled water using cellulose membranes (MW cut-off value of 12 kDa) for 3 days with frequent change of water (four times in 1 day). The purified copolymer solution was lyophilized, freeze-dried, and analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy for its final composition. Instrumentation and Methods. NMR Spectroscopy. Bruker DPX 400/200 and 100 MHz NMR spectrometer were used to record 1H NMR and 13C NMR spectra, respectively, with the residual solvent signal being used as an internal standard. The temperature-dependent 1H NMR spectra measurement was carried out with polymer solution (1 mM) in deuterium oxide (D2O) at a heating rate of 0.5 °C min−1, and the sample was equilibrated for 5 min after reaching

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EXPERIMENTAL SECTION Materials and Sample Preparation. Coumarin-153 (C153), rhodamine 6G (R6G), and Nile Red (laser grade, Exciton) were used as received. ((3-Methacrylamidopropyl)trimethylammonium chloride) (MAPTAC; 50 wt % solution in water), 4,4′-azobis (4-cyanovaleric acid) (ACVA), and doxorubicin hydrochloride were purchased from Sigma-Aldrich and used as received. N-Isopropylacrylamide (NIPA, SigmaAldrich) was recrystallized twice from hexane before use. S-1Dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate (DDMAT) was synthesized according to the procedure reported in literature with some modification.35,36 Details of the synthesis procedure of DDMAT and its 1H NMR and 13C 2275



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of water to get a series of solution with varying polymer concentration (0 to 0.02 mM) in which dye concentration remained constant. After sonication of 5 min, each vial was allowed to stand for 3 h, and emission spectra were recorded at an excitation wavelength of 550 nm while monitoring the emission from 570 to 800 nm. Emission intensity at 620 nm was plotted against concentration of polymer. The observed inflection point of this plot was considered as CAC. Dye Encapsulation. To investigate the dye-encapsulation ability of the block copolymer vesicles, we utilized R6G as a hydrophilic dye. A mixture of R6G (0.5 mM) and polymer (1 mM) in water at 37 °C was sonicated for 1 h, followed by dialysis against water through 3000 Da MWCO membrane for 48 h while removing outside water every 3 h interval to remove any nonencapsulated free dye molecule. After that, dyeencapsulated vesicular solution was investigated by employing UV and fluorescence spectroscopy. To quantify entrapped R6G, an absorbance spectrum of the dialyzed solution was recorded, and the absorbance obtained at 527 nm was matched with an aqueous R6G solution of known concentration, without any polymer. Concentration-normalized emission spectra of these two solutions were compared to establish the selfquenching phenomena of vesicle-encapsulated R6G dye. Doxorubicin Encapsulation. Prior to use, a weighed amount doxorubicin hydrochloride (1 mg/mL) in water was neutralized using a stoichiometric amount of 10 mM sodium hydroxide. The neutralized drug solution was freeze-dried, redissolved in ethanol, and then filtered through a membrane filter with a pore size of 0.2 μm to remove the precipitated salt. The resulting neutral doxorubicin (DOX) solution in ethanol was then used as a stock solution. A mixture of DOX (0.5 mM) and the block copolymer (1 mM) in phosphate buffer (pH 7.4) at 37 °C was gently stirred for 1 h, and the DOX-loaded vesicles were centrifuged at 37 °C, followed by dialysis against buffer solution (pH 7.4) through 3000 Da MWCO membrane for 4 h while removing of outside buffer solution at every 30 min interval to ensure complete removal of free drug molecule from the polymer solution. The amount of DOX released was determined by measuring its fluorescence intensity at 593 nm (λex = 480 nm).40 Fluorescence Quantum Yield Calculation. Fluorescence quantum yields of donor molecules have been calculated by using ethanolic solution of R6G (quantum yield 0.95)41 as a standard for the fluorescence quantum yield measurement according to the following eq 1

the desired temperature before recording the spectrum. The spectrum was recorded at 5 °C intervals in the temperature range of 25 to 50 °C. Turbidity Measurement. Aqueous polymer solution (1 mM) was filtered directly into the cuvettes through a membrane filter with a pore size of 0.2 μm and used for turbidity measurements. Turbidity measurement was done by monitoring the transmittance at 500 nm by using a Cary 5000 UV−vis-NIR spectrophotometer (Varian Scientific Instruments) fitted with a digital temperature controller. The solution was heated from 20 to 50 °C, and transmission was recorded. % Transmittance versus temperature plot was used to estimate the cloud point of the block copolymer solution. Size Distribution Measurement. The hydrodynamic diameter (Dh) of the aqueous polymer solution was obtained from dynamic light scattering (DLS) measurement, which was performed using a Malvern Nano ZS instrument well-appointed with a thermostated sample chamber by employing a 4 mW He−Ne laser (λ = 632.8 nm). In this instrument setup, the detector angle was fixed at 173°. Transmission Electron Microscopy. For obtaining transmission electron microscopy (TEM) micrographs, a negative staining technique was used in which a certain amount of aqueous polymer solution (0.1 mM) was sonicated for 1 h; then, 10 μL of this solution was allowed to settle for 2 min on a 300 mesh size carbon-coated copper grid (50 nm carbon film). Excess sample was sponged with a filter paper and dried, and a drop of freshly prepared aqueous 1% uranyl acetate solution was allowed to come in contact for 2 min. Throughout the previously described process, the temperature was kept constant (37 °C). The specimen was air-dried overnight and scanned using a transmission electron microscope (JEOL-JEM 2100, Japan) operating at an accelerating voltage of 80 kV at room temperature (25 °C). Steady-State and Time-Resolved Fluorescence Measurement. The absorption and fluorescence spectra were collected using a Shimadzu (model number, UV-2450) spectrophotometer and a JobinYvon - Spex Fluorolog-3 spectrofluorimeter, respectively, equipped with temperature-controlled watercooled cuvette holder. For steady-state experiments, all samples were excited at 408 nm. For time-resolved fluorescence measurements, we have used a time-correlated single photon counting (TCSPC) instrument from IBH, U.K. The instrument response function of this setup was ∼0.09 ns. The detailed time-resolved fluorescence setup is described by Hazra et al.37,38 In brief, the samples were excited at 408 nm using a picosecond laser diode (IBH, U. K. Nanoled), and the signals were collected at the magic angle (54.7°) using a Hamamatsu microchannel plate photomultiplier tube (3809U). The data analysis was performed using IBH DAS version 6 decay analysis software. All of the long and short wavelength decays were fitted biexponentially by considering that χ2 becomes close to 1, indicating a good fit. The temperature was kept constant (37 °C) by circulating water through the cell holder using a JEIO TECH thermostat (RW-0525GS). Determination of Critical Aggregation Concentration. Critical aggregation concentration (CAC) of the block polymer was estimated by using Nile Red as a fluorescence probe.39 A measured amount (10 μL) of stock solution of Nile Red in methanol (1.83 mM) was placed in different vials, and the solvent was evaporated. To each of these vials, varying amounts of aqueous polymer solution (0.5 mM) were added; then, the final volume (2 mL) was adjusted with an appropriate amount

2 2 Φsample = Φstd.[(I /A)sample × (A /I )std. ](ηsample /ηstd. )

(1)

where Φ represents quantum yield, A is absorbance at the excitation wavelength, η is the refractive index, and I is the integrated emission intensity calculated from the area under the emission peak. Calculation of FRET Parameters. According to the Förster theory, the rate expression for FRET is given by the following eq 2

kET =

6 1 ⎛ R0 ⎞ ⎜ ⎟ τD0 ⎝ r ⎠

(2)

where τ0D is the lifetime of the donor in the absence of acceptor, r is the distance between the molecular centers of the donor and acceptor, and R0 is called Förster distance, at which the efficiency of energy transfer is assumed to be 50%. The Förster distance can be calculated from the following eq 3 2276



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Figure 1. (a) Plot of transmittance (%) at 500 nm of 0.1 mM PNIPA-b-PMAPTAC and PNIPA homopolymer. (b) Plot of fluorescence emission intensity of Nile Red at 620 nm as a function of polymer concentration in water to determine the critical aggregation concentration (CAC).

R 06 =

9000(In10)κ 2 ΦD 128Π5Nn 4

∫0

∞

FD(λ)εA (λ)λ 4 dλ

b-PMAPTAC was investigated by monitoring optical transmittance at 500 nm with increasing solution temperature. The polymer solution was transparent below 33 °C, and the transparency started decreasing above this temperature (Figure 1a). However, the decrease in transmittance was less sharp compared with that for a PNIPA homopolymer, which generally exhibits an LCST at 32 °C,43,44 possibly due to the presence of highly hydrophilic PMAPTAC block. The cloud point of PNIPA block of PNIPA-b-PMAPTAC in water was found to be 36 °C, where 50% drop in transmittance was observed (Figure 1a). Because of this transition of PNIPA block from hydrophilic to hydrophobic above the cloud point, PNIPA-b-PMAPTAC was rendered amphiphilic above 36 °C. The solution was kept at 37 °C for over 2 weeks, and no precipitation was observed, indicating the soluble nature of the block copolymer at that temperature, which hints at the formation of stable self-assembled nanostructures by the amphiphilic PNIPA-b-PMAPTAC block copolymers. It is known that amphiphilic polymers can self-assemble and form stable nanostructures in water beyond a particular concentration, known as CAC.45 We have determined CAC of PNIPA-b-PMAPTAC using Nile Red as fluorescent probe.39 Aqueous solution of Nile Red showed very weak fluorescence, with emission maxima at 661 nm (λex = 550 nm). Figure 1b shows the plot of emission intensity of Nile Red as a function of polymer concentration in water. Nile Red emission intensity increased in a nonlinear manner as a function of polymer concentration. At low concentration range, no substantial change of Nile Red emission intensity or emission maxima was observed, which indicates that Nile Red remained in an environment similar to water. Beyond a particular concentration of the polymer, the emission intensity of Nile Red increased dramatically with simultaneous blue shifting of the emission maxima, which indicates the encapsulation of Nile Red in the hydrophobic shell produced by the aggregation of the block polymer molecules. A plot of Nile Red emission intensity as a function of polymer concentration produced an inflection point, and the concentration corresponding to that point was considered as the CAC of the block polymer in water. For the block copolymer used in the present study, the value of the CAC was found to be ∼0.008 mM or 0.27 mg mL−1 (Figure 1b), which is comparable to similar copolymers reported in the literature.12,45,46 To confirm that the self-assembled nanostructures were indeed formed, 1H NMR spectra of an aqueous solution of

(3)

where ΦD is the fluorescence quantum yield of the donor in the absence of the acceptor, κ2 is the orientation factor of two dipoles interacting, n is the refractive index of the medium, FD(λ) is the normalized fluorescence intensity of the donor when the acceptor is absent, that is, ∫ ∞ 0 FD(λ) dλ = 1, εA(λ) is the molar absorption coefficient of the acceptor, which is typically represented in mol−1 cm−1, and N is Avogadro’s number.

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RESULTS AND DISCUSSION

Synthesis of the Block Copolymers. Cationic block copolymers of MAPTAC and NIPA were synthesized by RAFT polymerization technique, as shown in Scheme 1. In the first step, PNIPA macro-CTA was synthesized by polymerizing NIPA in the presence of DDMAT CTA. The composition and number-average molecular weight of the synthesized polymers were determined by the utilization of 1H NMR spectroscopy because this method provides absolute and more reliable data for quantification.42 The molecular weight of the PNIPA macro-CTA was quantified by integration of the three protons of the terminal methyl group of dodecyl unit of DDMAT (at 0.88 ppm) with respect to one proton at 4.1 ppm of NIPA repeat units. (For 1H NMR spectra, please see the Supporting Information, Figure S3.) The PNIPA macro-CTA was found to consist of 100 NIPA units that correspond to a Mn of 11 300 g mol−1. In the next step, block copolymer with desired number of MAPTAC repeat unit was synthesized by polymerizing MAPTAC in the presence of PNIPA macro-CTA. The composition of the block copolymer was determined by integration of the intensities of the nine methyl protons adjacent to the quaternary nitrogen plus two methylene protons adjacent to amide nitrogen in the PMAPTAC block (at 3.32 ppm) and comparing with the intensity of one tertiary proton of isopropyl group in the PNIPA block (at 4.06 ppm) (Figure S4, Supporting Information). The molecular weight of the second block, PMAPTAC, was determined from the copolymer composition. The molar ratio of NIPA/MAPTAC was found to be 100:82, and the molecular weight of the block copolymer was calculated to be ∼29 400 g mol−1 with the PMAPTAC block having Mn of 18 100 g mol−1. Thermally Induced Self-Assembly of the Block Copolymer. Thermally induced phase transition of PNIPA2277



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Figure 2. 1H NMR spectra of 0.1 mM PNIPA-b-PMAPTAC in D2O with increasing temperature.

Figure 3. (a) Size distribution profile of the PNIPA-b-PMPTAC obtained from dynamic light scattering measurement at two different temperatures − below and above the cloud point. (b) TEM images of the vesicles obtained at 37 °C (scale bar = 500 nm) and image of a vesicle with higher magnification is shown in the inset (scale bar = 100 nm).

Figure 4. (a) Concentration-normalized fluorescence spectra of R6G encapsulated in 0.1 mM vesicular solution (red line) and in polymer-free water (black line) at 37 °C. (b) Intensity-normalized emission spectra of R6G encapsulated in vesicular solution at various time intervals. In both cases, the intensity was normalized with respect to the intensity of absorbance matched R6G solution at 557 nm in the absence of polymer.

PNIPA-b-PMAPTAC (0.1 mM) were recorded at various temperatures (25 to 50 °C), as depicted in Figure 2. The result was quite consistent with those obtained from turbidity measurements. At temperature ∼25 °C, the peaks corresponding to PNIPA and PMAPTAC blocks are all clearly visible. When the temperature of the polymer solution reached near the cloud point, the intensity of the peaks corresponding to the

PNIPA block started decreasing and completely disappeared on increasing the solution temperature above cloud point. Isopropyl groups of PNIPA are known to become dehydrated above its cloud point;47 therefore, the PNIPA block in the copolymer forms a hydrophobic domain above the cloud point and undergoes transition from molecularly hydrated unimers to aggregates. 2278



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Figure 5. (a) Fluorescence emission spectra displaying uptake of Nile red (λex = 550 nm) within PNIPA-b-PMAPTAC at 37 °C and subsequent release at 25 °C due to disassembly in aqueous medium. (b) Decrease in relative intensity against time shows the kinetics of burst release; control experiment was done to monitor any possible photobleaching of Nile Red under similar conditions.

Dye and Drug Release. As shown in the previous section, the CAC measurement also revealed the capability of the PNIPA-b-PMAPTAC block copolymers to encapsulate hydrophobic dye molecules Nile Red in the hydrophobic shell region, as revealed by substantial increase in the emission intensity with concomitant blue shift of ∼40 nm compared with its solution in pure water. We also studied thermally triggered release kinetics of the encapsulated dye molecules from the block copolymer vesicles by suddenly decreasing the temperature of the polymer solution to room temperature (25 °C) and simultaneously measuring the fluorescence intensity as a function of time (Figure 5a,b). The release of Nile Red from the hydrophobic shell of the vesicles to the bulk water on disintegration of the vesicles resulted in significant reduction in the intensity of the fluorescence of the dye. Fluorescence measurements as a function of time also provided an indication of the kinetics of the disassembly process (Figure 5b). It was concluded that most of the Nile Red was released during the first 30 min of the disassembly process, although we understand that this time may vary depending upon the cooling rate. We have also studied temperature-controlled release behavior of a chemotherapeutic drug DOX. DOX was encapsulated in the polymer vesicles at 37 °C, and its release behavior was studied at two different temperatures: one above (37 °C) and one below (25 °C) the LCST of the PNIPA block. At 37 °C, no detectable amount of DOX was released from the vesicles for up to 240 min, while on cooling the vesicular solution to 25 °C, the PNIPA cores within the vesicles transformed from hydrophobic to hydrophilic causing general rupture of the vesicles, thus triggering release of DOX (Figure S6, Supporting Information). It was found that considerable amount of DOX remained in the dialysis bag at 25 °C even after 240 min, which could be due to the interactions between uncharged DOX and the hydrophilic PNIPA chains at room temperature.13 However the ability of this vesicle to release drug molecules preferentially at temperatures below the LCST of PNIPA is proven by this experiment. Steady-State and Time Resolved Studies of FRET Process. To form the FRET donor−acceptor pairs within a vesicle, we utilized the amphiphilic nature of the vesicle to incorporate a hydrophobic fluorescent dye C-153 in the hydrophobic shell of the vesicles and R6G in the inside core and outside corona region of the vesicles. With both the donor (C-153) and acceptor (R6G) fluorescent dyes incorporated

DLS study was effectively used to study this thermally induced self-assembly of PNIPA-b-PMAPTAC. Figure 3a shows the DLS data at two representative temperatures, one below (25 °C) and one above (37 °C) the cloud point of PNIPA. The copolymer at 25 °C existed as hydrated unimers with intensity average hydrodynamic diameter (Dh) of 5.3 nm. At temperatures close to the cloud point, the unimers started self-assembling until above the cloud point where nanoparticles of ∼163 nm were formed. TEM of the polymer solution was performed at 37 °C for better understanding of the nature of the self-assembled nanostructures. TEM studies revealed the formation of spherical aggregates of diameter ∼250 ± 50 nm with a dimer wall thickness of ∼7 nm and a hollow interior, which is symptomatic of vesicular assembly (Figure 3b). The particle size obtained from TEM was higher than obtained from the DLS measurement, which can be explained in terms of flattening of vesicle during adsorption onto the TEM grid. Similar observation was reported previously as well.48 Dye Encapsulation. To further ascertain the formation of vesicular assemblies, these aggregates were generated in the presence of a hydrophilic dye, R6G. R6G was incorporated into the nanostructures by sonication of the aqueous solution of the block copolymer above the cloud point, followed by extensive dialysis to ensure of the complete removal of the nonencapsulated or free dye molecules. Figure 4a shows the emission spectra of R6G in pure water and when encapsulated inside the polymer vesicles at 37 °C. Emission obtained at 557 nm from the dialyzed solution containing R6G encapsulated inside the vesicles was much less intense (∼65%) than the emission of absorbance matched aqueous R6G solution in the absence of the block polymer at same temperature. This observed quenching of fluorescence is attributed to the confinement of R6G into the water-filled interior of the vesicle. This self-quenching nature of the hydrophilic dye molecule due to confinement has been previously utilized to ascertain the formation of vesicular assembly.49,50 Subsequently, on decreasing the temperature of the above R6G encapsulated vesicular solution to room temperature (25 °C), the fluorescence intensity of the resulting solution became close to that of polymer-free solution at the same temperature. To check the stability of such vesicles at 37 °C, the fluorescence emission intensity of dialyzed R6G encapsulated vesicular solution was recorded at various time intervals. No substantial change of emission intensity demonstrates high kinetic stability of these polymer vesicles (Figure 4b). 2279



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Figure 6. (a) Overlap between emission spectra of C-153 and absorption spectra of R6G in 0.1 mM PNIPA-b-PMAPTAC solution in water at 37 °C. (b) Steady-state fluorescence spectra of only donor, acceptor, and acceptor + donor in the same aqueous polymer solution.

Figure 7. (a) Steady-state fluorescence quenching spectra of C-153 with varying R6G concentration in 0.1 mM PNIPA-b-PMAPTAC copolymer solution at 37 °C. (b) Time-resolved fluorescence decay of C-153 with varying concentration of R6G in the same aqueous polymer solution. The variation of the average lifetime of the donor as a function of acceptor concentration is shown in the inset.

intensity of C-153 (12.8 μM) in the presence and absence of acceptor (Figure 6b) and found a pronounced decrease in donor emission in the presence of the acceptor molecules. This clearly indicated the occurrence of effective FRET from the donor C-153 to the acceptor R6G. Additionally, it was also observed that upon stepwise addition of R6G in 0.1 mM aqueous polymer solution containing fixed amount of donor (C-153, 12.8 μM) the donor emission decreased gradually, as shown in Figure 7a. Information on some useful parameters, namely, spectral overlap integral [J(λ)] between the emission and absorption spectrum of the donor and acceptor, respectively, fluorescence quantum yield of donor in the absence of acceptor (Φsample), and the Forster distance (R0) between donor and acceptor in polymersomes were obtained from the steady-state studies and are listed in Table 1. Although steady-state measurement provided much useful information, further confirmatory results could be obtained from time-resolved analysis with picosecond setup. In timeresolved measurements, the energy transfer processes are

inside, these polymer vesicles may serve as the scaffolds for FRET process. Steady-state UV−vis absorption and fluorescence emission spectra of acceptor (R6G) and donor (C-153) were recorded at 37 °C in water and in 0.1 mM aqueous solution of the block copolymer. In water, the absorption maxima of R6G and the emission maxima of C-153 were found to be 526 and 550 nm, respectively, similar to the previously reported data,51 whereas, in the case of 0.1 mM block copolymer solution containing vesicles, the absorption maxima of R6G and the emission maxima of C-153 were found to be 527 and 525 nm, respectively. So, in vesicular environment, significant (25 nm) blue shift occurred in the emission maxima of C-153 along with significant increase in fluorescence intensity (Figure S5, Supporting Information), whereas no shift was observed in the absorption maxima of R6G. This indicated that the distribution of C-153 favored the hydrophobic bilayer region of the vesicles. The effect of polymersomes on donor emission caused a huge increase in the spectral overlap of the acceptor (R6G) and the donor (C-153) in comparison with pure water, making the FRET process more effective. The representative overlap of emission spectra of donor and absorption spectra of the acceptor in 0.1 mM aqueous polymer vesicles is shown in Figure 6a. With the addition of the donor (C-153, 12.8 μM) to a 0.1 mM aqueous vesicular solution containing acceptor (R6G) at two different concentrations, a significant increase in the fluorescence intensity of the acceptor was observed (Figure 6b). At the same time, we have also compared the emission

Table 1. FRET Parameters for C153-R6G Pair in Polymersome λex (nm)

J(λ) (M−1 cm−1 nm4)

R0 (Å)

Φsample

r (Å)a

kET (s−1)a

408

3.13 × 1015

50.24

0.41

53.38

1.86 × 108

a

2280

From time-resolved experiment, as discussed later. dx.doi.org/10.1021/jp412273h | J. Phys. Chem. B 2014, 118, 2274−2283


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observation is in agreement with the previous literature reports.52 The highest energy transfer efficiency value corresponds to the shortest donor−acceptor distance. Now the distance (r) between the molecular centers of the donor and acceptor can be determined from the energy transfer efficiencies from the following eq 6.

monitored by change in the donor lifetime with the addition of varying amount of acceptor. The results obtained in our system are shown in Figure 7b. The fluorescence lifetime was measured at the emission wavelength of 525 nm, that is, the emission maxima of C-153. With the addition of the acceptor, the donor lifetime decreased, as given in Table 2. The average lifetime for Table 2. Decay Parameters of C-153 with Varying R6G Concentration in Vesicle (λex = 408 nm)a

a

1+

donor (C-153) conc. (μM)

acceptor (R6G) conc. (μM)

τ1 (ns)

α1

τ2 (ns)

α2

(ns)

12.8 12.8 12.8 12.8 12.8

0.0 2.1 4.2 8.4 12.6

1.499 1.396 1.339 1.224 1.058

0.41 0.47 0.53 0.57 0.55

5.295 4.680 4.222 3.830 3.590

0.59 0.53 0.47 0.43 0.45

3.74 3.14 2.69 2.34 2.19

multiexponential fluorescence decay was determined by the following eq 4. The variation of the average lifetime of the donor as a function of acceptor concentration is shown in the inset of Figure 7b (4)

where τ1 and τ2 are the lifetime components with their corresponding relative weightage, α1 and α2. In a complex situation like the present one, where there is a distribution of donor−acceptor distances, we have used average lifetime quenching data to determine energy transfer efficiency, which is assumed to be more accurate than the steady-state fluorescence quenching data. In vesicular assemblies, variation of multiple donor−acceptor distances depends on the distribution of donor molecules inside the vesicle. The concentration of acceptors plays an important role because the donor−acceptor proximity is governed by the acceptor concentration. FRET efficiency (E) is calculated using the following eq 5 τ E = 1 − DA τD (5) where τDA and τD are the lifetimes of donor in the presence and absence of acceptor, respectively. Table 3 reflects the acceptor Table 3. FRET Efficiency of C-153 (12.8 μM) with Varying Concentration of R6G in Polymersomea

a

concentration of R6G (μM)

FRET efficiency (%) from time-resolved measurement

2.1 4.2 8.4 12.6

16 28 37 41

6

( ) r R0

(6)

In these polymer vesicles, we previously concluded that the donor molecules (C-153) were located solely within the hydrophobic bilayer, whereas the acceptor molecules (R6G) resided preferentially into the water-filled interior and exterior of the vesicles. The FRET study revealed that the shortest donor−acceptor (D−A) distance for the C-153−R6G pair in the present case is ∼53.4 Å. The size of the vesicles is much larger (as observed from the TEM images) than the D−A distance calculated from the FRET study, which indicates intravesicle FRET rather than intervesicles.53 Considering the location of the donor and the acceptor molecules, one can logically correlate the measured D−A distance with the thickness of vesicular bilayer if the FRET occurs within the same vesicular lumen. In the common surfactants (e.g., CTAB/ SDS) forming cationic unilamellar vesicles, it has been reported that the thickness of the bilayer is in the range 24−34 Å.54 It should be noted that the distances reported by Das et al.53 from the FRET studies in other vesicular systems correlated well with the thickness of the bilayer. In view of these points, the shortest D−A distances, as obtained from the FRET studies in the present study, provide useful information regarding the average thickness of the bilayer. The above results demonstrate that the present PNIPA-b-PMAPTAC vesicular system can provide a suitable scaffold for the FRET process. FRET-based stimuli-responsive nanocarriers for drug delivery are used to monitor the stimuli-responsive dynamic release of drugs in the biological system.25,55,56 When vesicles disassembled by the external stimuli, the FRET molecules were released and diffused apart, eliminating the energy transfer. By monitoring the FRET efficiency, release of the core-loaded probes to biological media can potentially be demonstrated. In summary, thermally responsive diblock copolymer PNIPA-b-PMAPTAC were successfully prepared by RAFT polymerization. At room temperature, these block copolymers exist as unimers in aqueous solution and self-assemble into vesicles at temperature ≥36 °C. These thermoresponsive and permanently cationic vesicles can encapsulate both hydrophobic and hydrophilic molecules. They can be easily disassembled by simply decreasing the temperature of the medium and, as a result, can release the encapsulated cargo. Thus, these block copolymeric vesicles can potentially be utilized as a temperature-responsive model drug and genedelivery vehicle. It was also demonstrated that the present PNIPA-b-PMAPTAC vesicular system could provide a suitable scaffold for the FRET process.

Error is ±10% in all TCSPC results.

⟨τ ⟩ = τ1α1 + τ2α2

1

E=

Error is ±10% in all TCSPC results.

concentration dependence of FRET efficiencies with a fixed donor concentration. For lower acceptor concentrations, that is, lower acceptor/donor ratios, steady increase in FRET efficiencies was observed with increasing acceptor concentration as the probability of finding a suitable energy acceptor in the proximity of the donor increases. This change of FRET efficiencies with acceptor concentration becomes saturated after reaching a particularly high acceptor/donor ratio. The above

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ASSOCIATED CONTENT

S Supporting Information *

Details of the synthesis procedure of DDMAT and its 1H NMR and 13C NMR spectra, 1H NMR spectra of PNIPA macro-CTA and PNIPA-b-PMAPTAC, and some more spectroscopic data. 2281



Article

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This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Tel: +91-3222-282326. Fax: +91-3222-282252. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Department of Science and Technology, Government of India, New Delhi (Project ref No. SR/FTP/CS-79/2007) is acknowledged. Research Fellowships to C.M. and D.D. from UGC, New Delhi, and to S.M. from CSIR, New Delhi, are also acknowledged.

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Thermoregulated Formation and Disintegration of Cationic Block

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