Energy-Transfer Phenomena in Thermoresponsive and pH

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Energy Transfer Phenomena in Thermo-Responsive and pH-Switchable Fluorescent Diblock Copolymer Vesicles Chiranjit Maiti, and Dibakar Dhara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01891 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Energy Transfer Phenomena in Thermo-Responsive and pHSwitchable Fluorescent Diblock Copolymer Vesicles

Chiranjit Maiti and Dibakar Dhara* Department of Chemistry Indian Institute of Technology Kharagpur West Bengal 721302 India

ACS Paragon Plus Environment

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ABSTRACT We describe the development of a polymeric vesicle that not only selectively fluoresce at low pH, a condition prevailing in cancer cells, but also can potentially monitor thermoresponsive release of a drug even if the drug is non-fluorescent. The developed fluorescence resonance energy transfer (FRET)-based thermoresponsive vesicular nanocarriers comprised of a new poly(PEGMA)-b-poly(NIPA-r-R6GMED) block copolymer, which undergoes pH switchable superior turn ‘on-off’ fluorescence characteristics. The block copolymer was synthesized using RAFT technique, and its solution properties and self-assembly behavior were investigated by turbidity measurement, fluorescence spectroscopy, 1H NMR, dynamic light scattering and transmission electron microscopy. The block copolymer self-assembled to form nanostructured vesicles above critical aggregation temperature in physiologically relevant condition. Steadystate and time-resolved fluorescence spectroscopy were utilized to study FRET process between encapsulated hydrophobic guest C-153 (donor) and polymer-bound R6GMED units (acceptor) in the thermo-responsive vesicles. FRET rate and efficiency was found to vary due to pHdependent changes in quantum yield of the acceptor molecules. The occurrence of a highly efficient FRET in this polymeric vesicular nanocarrier in acidic pH, a condition similar to cytoplasm and cell nucleus in leukemic tissues, as well as the ability to encapsulate hydrophilic and hydrophobic molecules and their temperature controlled release make it potentially useful as imaging guided real-time monitoring drug delivery vehicles.

KEYWORDS: Polymersomes, Nanoparticles, RAFT, Fluorescence Spectroscopy, FRET, Thermoresponsive Drug Delivery, Cancer Chemotherapy.


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INTRODUCTION A great deal of effort has been recently focused on the development of effective drug delivery systems (DDS), taking advantage of the differential microenvironmental conditions offered by diseased or injured cells in comparison to physiologically normal cells and tissues.1-3 In this regard, nanostructures from stimuli-sensitive amphiphilic block copolymers have generated great potential in delivery of drugs and genetic materials.4-5 Among the various stimuli-responsive polymers, the area of thermoresponsive polymers is particularly interesting.6-7 Block copolymers containing thermoresponsive blocks that show lower critical solution temperature (LCST) in water are appealing as they can produce various self-assembled nanostructures such as micelles89

and vesicles10-11 as DDSs for therapeutics above a critical aggregation temperature (CAT).12

Polymer vesicles are usually spherical nanoparticles made by self-assembly of amphiphilic block copolymers with an aqueous core that is surrounded by a hydrophobic shell which enable loading of both hydrophobic and hydrophilic guest molecules in the vesicles.13 The encapsulated cargo can be released from the vesicles by triggering stimuli-induced disassembly of vesicles.14-15 Fluorescence resonance energy transfer (FRET) is a distance-dependent nonradiative energy transfer process from an excited fluorophore (the donor) to another fluorophore (the acceptor).16 The efficiency of FRET is inversely proportional to the sixth power of donor-to-acceptor separation that established it as a sensitive technique for investigating a variety of biological phenomena that produce changes in molecular proximity.17 Thus, to apply FRET process effectively, it is necessary to accurately control the relative positioning of the participating fluorophores. Accurate positioning of the fluorophores can be achieved by placing them in the various compartments of block copolymer nanoassemblies that enable the FRET process to occur.18-19 FRET technique was used to probe polymeric aggregates in various occasions e.g., to


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monitor micellar swelling, vesicle-to-micelle transition, polymer supramolecular self-assembled state, as well as encapsulation and release of drugs from DDSs induced by external/internal stimuli.20-23 Among the internal stimuli, pH is known to be very important because of its differential value between normal healthy cells and cancer cells.24 The lower pH value in cancer cells can be utilized to design smart optical probes that can selectively illuminate tumor cells which is essential for improved treatment of tumors.25 FRET can also potentially be used in development of strategies for real-time monitoring of drug release inside the targeted cells. But, non-fluorescent nature of most of the current drug candidates poses challenges in investigating the release of drug in complex cellular microenvironments. This limitation necessitates the development of a real-time monitoring system within the stimuli responsive DDSs. To address the above-mentioned concerns, in this work, we have utilized reversible additionfragmentation chain transfer (RAFT) process to synthesize a new poly(PEGMA)-b-poly(NIPA-rR6GMED) thermoresponsive block copolymer that spontaneously form vesicles above a certain temperature. We show that both hydrophilic and hydrophobic molecules can be encapsulated in this new vesicle which can be released by decreasing the temperature. Furthermore, R6GMED units that are incorporated randomly along with NIPA units into one of the blocks of the copolymer, undergo pH switchable reversible transformation from spirolactam to ring-opened amide form due to protonation26 and exhibits superior turn ‘on-off’ fluorescence characteristics. We have also studied FRET rate and efficiency between an encapsulated hydrophobic guest C153 and polymer-bound R6GMED units in the vesicles as a result of pH-dependent changes in quantum yield of the donor-acceptor molecules. Therefore, this FRET-based stimuli-responsive vesicle can certainly be helpful to demonstrate dynamic release of any drug molecules, without relying on the drugs' fluorescence properties, by monitoring effective intraoperative change in


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FRET parameters inside the tumor cells and hence, may lead to optimal clinical outcomes in cancer therapy.

EXPERIMENTAL SECTION Materials and Sample Preparation. Poly(ethylene glycol) monomethyl ether acrylate (PEGMA, MW 480), ethylenediamine, rhodamine 6G (R6G) were purchased from SigmaAldrich (St. Loius, MO, USA) and used as received. 2,2′-Azobisisobutyronitrile (AIBN) and Nisopropylacrylamide (NIPA) (Sigma-Aldrich) were purified by recrystallizing twice from methanol and hexane respectively. Methacryloyl chloride was purchased from Alfa Aesar (MA, USA) and redistilled whenever required as reagent. Coumarin-153 (C153), rhodamine B (RB) were purchased from Exciton (Dayton, OH, USA). N-(Rhodamine-6G)lactam-N'-methacryloyl ethylenediamine (R6GMED) and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPPA) were synthesized following a reported procedure with slight modifications.27-28 Detail synthesis procedures for R6GMED and CPPA and the 1H NMR and

13

C NMR spectra of all essential

compounds are presented in the Supporting Information (Figures S1 – S6). Aqueous solutions of a dye (RB or C153) were prepared by pipetting out required amount of the dye from a methanolic stock solution following which the methanol was evaporated carefully through nitrogen gas purging. Then, appropriate volume of polymer solution poured to the dye to achieve the required final dye and polymer concentration. Mlili-Q water was used for all experiments.

Synthesis of Poly(PEGMA)-Based Macro-Chain Transfer Agent (CTA) (Scheme 1). Synthesis of PPEGMA macro CTA was performed by the controlled polymerization of PEGMA in 1,4-dioxane using AIBN and CPPA as the initiator and CTA, respectively. At first, PEGMA


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Scheme 1: Synthetic scheme for diblock copolymer poly(PEGMA)-b-poly(NIPA-r-R6GMED) using RAFT polymerization technique.

(3.0 g, 6.2 mmol), CPPA (0.045 g, 0.16 mmol) and AIBN (0.006 g, 0.04 mmol) with monomer (PEGMA) to CTA ratio ( ⁄ ) of 40:1 and CTA to initiator ratio ( ⁄ ) of 4:1 were taken in a 10 mL single necked round-bottomed flask containing 2 mL 1,4-dioxane, magnetic stir bar and sealed by a septa. The reaction mixture was purged thoroughly with N2 gas for 30 minutes and then the mixture was stirred in oil bath at 70 °C for 16 h following which the reaction flask was dipped in a liquid nitrogen bath to quench the polymerization reaction. Methanol was added to the reaction mixture for dilution following which the resulting mixture was dialyzed for 24 h against methanol using a dialysis bag (MW cut-off = 12 kDa). Outside methanol was replaced in every 6 h. The product was obtained by evaporating the methanol from the dialyzed mixture which was further dried overnight under high vacuum. The composition and number average molecular weight analysis of the polymer was carried out by 1H NMR spectroscopy and gel permeation chromatography (GPC) (Figure S7, 1 and S8).


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Synthesis of Diblock Copolymer - Poly(PEGMA)-b-Poly(NIPA-r-R6GMED) (Scheme 1). Block copolymer of PEGMA, NIPA and R6GMED was synthesized by performing radical polymerization of the mixture of NIPA and R6GMED in a desired mol ratio in presence of desired amount of poly(PEGMA) macro-CTA. The polymerization was carried out in dry N,Ndimethylformamide (DMF) using AIBN as the initiator at 70 °C. We have taken NIPA (0.38 g, 3.4 mmol) and R6GMED (0.26 g, 0.50 mmol) in ~135:20 mol ratio in a single necked roundbottomed flask sealed by a septa to which poly(PEGMA) macro-CTA (0.4 g, 0.024 mmol) was added making monomer to CTA ratio ( ⁄ ) of 155:1 while the molar ratios of ⁄ was kept constant at 4:1. The reaction mixture was purged with

nitrogen for 30 min and kept in a preheated oil bath at 70 °C with constant stirring. The polymerization was quenched after 24 h by dipping the reaction flask in liquid nitrogen. Then, DMF was added in the reaction mixture for dilution and the product was obtained by precipitation from a 1:1 (v/v) ice-cold mixture of diethyl ether and hexane. The precipitation process was repeated three times and solid product was then vacuum dried for 12 h. The molecular weight and composition of the synthesized diblock copolymer was analyzed by 1H NMR spectroscopy and GPC (Figures 1, 2 and S8). Instrumentation and Methods NMR Spectroscopy. Bruker Avance II 400/ Bruker Ascend 600 MHz and 100/150 MHz NMR spectrophotometers were used to record 1H NMR and

13

C NMR spectra of the samples. The

residual solvent peak was utilized as an internal standard. Temperature dependent 1H NMR spectra measurement was recorded using 0.08 mM polymer solution in D2O at a fixed solution pH by applying a heating rate of 0.5 °C min-1. After reaching the desired temperature, the sample


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was incubated for 5 min before recording a spectrum. The spectra were recorded in the interval of ~ 5-7 °C between 20 °C to 37 °C. Gel Permeation Chromatography (GPC). The molecular weight and dispersity (Ð) of the polymers were determined by a triple detector GPC, Viscotek TDAmax system equipped with refractive index, differential pressure viscometry and dual-angle light scattering (λ = 670 nm, 90° and 7°) detectors and an Agilent 1200 model isocratic pump, using tetrahydrofuran (THF) as mobile phase with a flow rate of 1 mL min-1 at 33 °C. Use of three detectors enabled us to determine absolute molecular weight of the experimental polymers. dn/dc values of the synthesized polymers were determined from the RI signals of four solutions of different concentration of each polymer and assuming 100% recovery of the polymers. The detector signals were calibrated using a polystyrene standard having narrow molecular weight distribution with known refractive index increment and intrinsic viscosity values (Mn = 105164, Mw/Mn = 1.02, [η] = 0.48 dL g-1 at 33 °C in THF, dn/dc = 0.185 mL g-1, provided by the supplier). Turbidity Measurement. Aqueous solutions of the block copolymer (0.08 mM) with different values of pH were taken in cuvettes at 10 °C and then turbidity was determined by measuring the transmittance at 625 nm where there is no absorption from any of the polymer components by using a Shimadzu (model no. UV-2405) spectrophotometer. The solution temperature was increased from 20 °C to 45 °C and % transmittance was plotted against solution temperature at different solution pH to evaluate the critical aggregation temperature (CAT) of the block copolymer. Determination of Critical Aggregation Concentration. Critical aggregation concentration (CAC) of the copolymer was determined by fluorescence spectroscopy using Nile Red as a hydrophobic probe.29 For fluorescence measurement, 15 µL of methanolic Nile Red solution (1.83 mM) was


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added in vials and mathanol was removed by evaporation. To these individual vials, measured volumes of aqueous polymer solution (0.3 mg/mL) were added and the volumes were made up to 1 mL with phosphate buffer (PB) of pH 7.4 to obtain a series of solutions with varying concentration of polymer (0 to 0.01 mM) and fixed Nile Red concentration. The solutions was sonicated for 5 min, allowed to stand for 3 h before recording the fluorescence spectra with λex of 550 nm using JobinYvon - Spex Fluorolog-3. The CAC was determined from the inflection point of the plot of emission intensity at maxima against concentration of the polymer. Size Distribution and Structural Characterization. Dynamic light scattering (DLS) measurements were performed to determine the hydrodynamic size distribution of polymer aggregates using a Malvern Nano ZS instrument having a temperature-controlled sample chamber by utilizing a 4 mW He-Ne laser (λ = 632.8 nm). Scattering photons were collected at a fixed detector angle of 173° and scattering intensity obtained from each sample was processed by instrumental software which provide the hydrodynamic diameter ( ). In DLS measurements, the water used for preparation of polymer solutions was first filtered through a membrane filter (pore size of 0.2 µm) to make the solutions dust-free. Transmission electron microscopy (TEM) measurements were performed for structural analysis of self-assembled polymer nanoparticles. The polymer solution (0.08 mM) was sonicated for 1 h and then 10 µL of this solution was drop casted on carbon-coated copper grid (300 mesh size, 50 nm carbon film) for 2 min. Excess solution was removed by a filter paper. The temperature was kept constant at 37 ⁰C throughout the sample preparation process. The specimen was dried for overnight and TEM micrographs were recorded using a transmission electron microscope (JEOL-JEM 2100, Japan) at room temperature (25 °C) operating at an accelerating voltage of 200 kV. Cryo-TEM measurements were performed using the same instrument as mentioned above; the sample was prepared by


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blotting a drop of the sample on a carbon coated Cu grid and then plunging the sample grid into liquid ethane. The specimen grid was kept under liquid nitrogen and finally mounted into the cryo-TEM holder. Steady-State and Time Resolved Fluorescence Measurement: Steady-state fluorescence spectra were collected using JobinYvon - Spex Fluorolog-3 and Hitachi (model No. F-7000) spectroﬂuorimeter by employing a 1 cm path length quartz cuvette. For time resolved fluorescence intensity decay measurements, a time correlated single photon counting (TCSPC) instrument (IBH, UK) was used. The instrument response function (IRF) was ∼ 90 ps in this setup. The details about this instrument setup is described elsewhere by Hazra et al.30-31 Briefly, a picosecond pulsed laser (IBH, UK. Nanoled) was used to excite the samples at 408 nm, and a Hamamatsu micro-channel plate photomultiplier tube (3809U) was utilized to collect the emission signals at the magic angle (54.7°). The data were analyzed using IBH DAS (version 6) software. The decays were fitted exponentially and χ2 values close to 1 showed a good fit. The temperature was maintained at 37 ⁰C during the entire measurement. Calculation of Fluorescence Quantum Yield: Fluorescence quantum yield of donor (C-153) molecules encapsulated into the block copolymer nanostructures was calculated by using Coumarin 480 as a secondary standard (reported quantum yield is 0.66 in water32) according to the following equation (1), Ф = Ф. !" #$

× "# $

.

&'

) ( *) + (.

(1)

where Ф represents quantum yield, I, A and η are emission intensity, absorbance at the excitation wavelength and refractive index respectively.


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Encapsulation of Rhodamine B (RB) in the Polymer Nanostructures: In order to show the ability of the block copolymer nanostructures to encapsulate hydrophilic dye molecules, RB was employed as a hydrophilic dye. RB (0.32 mM) and the copolymer (0.88 mM) were added in phosphate buffer (pH 7.4, 10 mM) at 10 °C and stirred well. Then the solution temperature was increased to 37 °C, sonicated for 30 min and dialyzed against PB solution of pH 7.4 through 12000 Da MWCO membrane for 24 h with replacing of outside buffer in every 4 h interval to eliminate any non-encapsulated free dye molecules. After RB was encapsulated in copolymer nanostructures, solutions were examined by using UV-vis and fluorescence spectroscopy. The absorbance maxima of the dialyzed solution containing encapsulated RB was obtained at 553 nm. A RB solution in phosphate buffer, without any polymer, was prepared to match the absorbance value of the encapsulated RB at 37 °C, pH 7.4.

RESULTS AND DISCUSSIONS Synthesis of the Block Copolymer - Poly(PEGMA)-b-Poly(NIPA-r-R6GMED): Well-defined block copolymer from PEGMA, NIPA and R6GMED was synthesized as shown in Scheme 1 by RAFT technique, which is one of the most efficient methods for synthesis of block copolymer. At first, poly(PEGMA) macro-CTA was synthesized by polymerization of PEGMA in dioxane using CPPA as chain transfer agent (CTA). The absolute molecular weight of the as synthesized macro-CTA was determined by end-group analysis using 1H NMR spectroscopy as well as GPC. End-group analysis for molecular weight quantification of poly(PEGMA) macro-CTA was done by relating integration values of the five protons of the phenyl group of CPPA from 7.62 to 8.10 ppm and of the three protons of terminal methyl group of PEGMA repeat units at 3.41 ppm (Figure S7, Supporting Information). The number-average molecular weight (Mn) of


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poly(PEGMA) macro-CTA was found to be ∼16100 g mol-1 corresponding to 33 PEGMA units. GPC analysis of this macro-CTA (Figures 1, S8) revealed narrow disperse polymer with an absolute Mn value of ∼16700 g mol-1 and dispersity (Ð) value of 1.21, indicating a good corroboration of Mn values obtained between the two techniques.

Figure 1: The molecular weight distribution by GPC of poly(PEGMA) macro-CTA and the diblock copolymer, poly(PEGMA)-b-poly(NIPA-r-R6GMED). In the next step, the synthesized poly(PEGMA) macro-CTA was employed for the preparation of random block copolymer of NIPA and R6GMED by carrying out polymerization reaction at a desired ratio of these two different monomers. The copolymer composition of the resulting poly(PEGMA)-b-poly(NIPA-r-R6GMED) block copolymer was determined by comparing the integration value of one aromatic proton of R6GMED units at 6.88 ppm as well as of the one tertiary proton of isopropyl group at 3.84 ppm coming from NIPA units of the second block with the intensities of three protons of terminal methyl group at 3.22 ppm in the poly(PEGMA) block (Figure 2). The molar ratio of NIPA:R6GMED in the second block was found to be approximately equal to 10:1 and the Mn of the block copolymer was found to be ∼36800 g mol-1 with the second block having Mn of 20700 g mol-1. Furthermore, the absolute Mn value was also determined by GPC


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Figure 2: 1HNMR spectra (in DMSO-d6) of diblock copolymer, poly(PEGMA)-b-poly(NIPA-rR6GMED). analysis as ∼37700 g mol-1, which is very close to the data obtained from end group analysis from 1H NMR spectroscopy. The molecular weight distribution was determined by GPC and the Ð value was found to be 1.34 (Figures 1, S8), confirming the successful controlled polymerization of the monomers. Detailed polymer characterization results are summarized in Table 1. Table 1: Results from the RAFT polymerization of NIPA and R6GMED in the presence of poly(PEGMA) macro-CTA in dry N,N-dimethylformamide (DMF) at 70 °C. Polymer

Mn,NMRa (gmol-1)

Mn,GPCb (gmol-1)

Mw,GPCb Mz,GPCb (gmol-1) (gmol-1)

Poly(PEGMA) macro-CTA Poly(PEGMA)-b-Poly(NIPA-r-R6GMED)

16,100 36,800

16,700 37,700

20,100 50,500

a

Calculated from 1H NMR spectroscopy. bObtained from GPC.


23,700 69,700

Ðb 1.21 1.34

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Thermally Induced Self Assembly of the pH-Switchable Fluorescent Block Copolymer: After successful synthesis of the diblock copolymer, poly(PEGMA)-b-poly(NIPA-r-R6GMED), in which one of the blocks consists of highly hydrophilic PEGMA repeat units and the other block consists of random distribution of NIPA and R6GMED repeat units, we have studied selfassembly and solution properties of the block copolymer by the utilization of turbidity measurement, fluorescence spectroscopy, DLS, TEM, and 1H NMR techniques. To study the thermoresponsive solution properties of poly(PEGMA)-b-poly(NIPA-r-R6GMED), the polymer was solubilized directly in PB (pH 7.4, 10 mM) at a concentration of ~ 0.1 mM which is significantly higher compared to the critical aggregation concentration (CAC) of similar copolymers reported in literature.33-35 Figure 3a shows that, at pH 7.4, the block copolymer solution was transparent below 20 °C, transmittance values started decreasing with increasing solution temperature and found to be almost invariant after 30 °C. It is known that the PNIPA homopolymer shows a lower critical solution temperature (LCST) of 33 °C in water36-37, beyond this temperature the transparency of PNIPA solution decreases sharply and reaches nearly zero.33 However, the lowest transmittance value for the present copolymer solution was ~ 40 % and the polymer did not precipitate out from the solution suggesting formation of some kind of selfassembled nanostructures. The critical aggregation temperature (CAT), a temperature above which the aggregation takes place, was considered to be 25 °C at pH 7.4, corresponding to 50 % drop in transmittance. This value of CAT is significantly less than the LCST (32 °C) of polymer containing only NIPA units which can be attributed to the random incorporation of hydrophobic R6GMED repeat units into the PNIPA block of the copolymer. Similar observation has been reported earlier by Kim et al. with random incorporation of hydrophobic comonomers into the PNIPA chain.38 We have also determined the CAC of the block copolymer using Nile Red as


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fluorescent probe.29 Figure S9 shows the Nile Red fluorescence intensity in polymer solutions of varying concentration in pH 7.4 at 37 °C, which is above the CAT as discussed earlier. The CAC of the block polymer in water was considered to be the concentration corresponding to the inflection point which was found to be 0.002 mM.

Figure 3: (a) Plot of transmittance (%) at 625 nm of 0.88 mM poly(PEGMA)-b-poly(NIPA-rR6GMED) copolymer solution at two representative pH values, pH 7.4 and pH 4.2. (b) Fluorescence emission spectra (excitation at ,- = 480 nm) of 0.88 mM poly(PEGMA)-bpoly(NIPA-r-R6GMED) copolymer as a function of solution pH below at 20 °C.

Poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer was amphiphilic above 25 °C because of the transition of poly(NIPA-r-R6GMED) block to hydrophobic from hydrophilic above this temperature. Furthermore, R6GMED units present in the poly(NIPA-r-R6GMED) block in the copolymer contains spirolactam ring, whose stability depends upon the environmental pH.39-40 We have investigated fluorescence properties of poly(PEGMA)-b-poly(NIPA-r-R6GMED) by gradually decreasing the solution pH from 9 to 3 at 20 °C (shown in Figure 3b). No significant fluorescence was observed at neutral and alkaline pH (7 and above) but on lowering solution pH (pH ≤ 6), a significant increase of fluorescence intensity was detected with emission maxima at


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558 nm (,- = 480 nm). In alkaline pH, the predominant stabilization of non-fluorescent spirolactam form of the R6GMED units present in the copolymer takes place, whereas under acidic condition (pH ≤ 6), highly fluorescent ring-opened amide form of the R6GMED units was preferred over spirolactam in presence of hydrogen ion. A schematic representation of this spirolactam / ring-opened amide form of R6GMED units present in the copolymer has been shown in Figure S10 (Supporting Information). Now, in order to see whether this pH- switchable reversible transformation of spirolactam structure to ring-opened amide form of R6GMED units present in the copolymer has any effect on copolymer self-assembly, we have also performed turbidity measurement by lowering the solution pH from 7.4 to 4.2 (Figure 3a). Slight increase of the CAT of ~ 1.5 °C at solution pH of 4.2 compared to pH 7.4 could be due to slight decrease of hydrophobicity of the R6GMED units in which the ring-opened amide form predominated. The block copolymer solution was kept in a vial for over 2 weeks at 37 °C in both the pH 7.4 and 4.2. Slight increase in hydrodynamic size with no precipitation suggested the formation of reasonably stable nanostructures by the self-assembly of amphiphilic poly(PEGMA)-b-poly(NIPA-rR6GMED) in physiologically relevant condition (please see Figure S11 in SI for the data on time-dependent size of the copolymer nanostructures). To confirm the formation of self-assembled nanostructures, 1H NMR spectra of 0.88 mM block copolymer solution was recorded in deuterium oxide at pH 7.4 in the temperature range between 20 °C to 37 °C) (Figure 4a; please refer Figure S12 in SI for the full spectra). The results corroborated the findings from turbidity measurements. The peaks corresponding to NIPA and PEGMA units are all prominent in spectra at temperature ~ 20 °C, but on increasing the temperature, attenuation of the characteristic peaks corresponding to NIPA units was observed which nearly disappeared above CAT. This observation signifies dehydration of the isopropyl


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groups of NIPA units above CAT.41 As discussed before, the random presence of few hydrophobic R6GMED units in the poly(NIPA-r-R6GMED) block of the copolymer decreases the cloud point of the particular block to ~ 25 °C compared to the cloud point of PNIPA

Figure 4: (a) 1H NMR spectra of diblock copolymer poly(PEGMA)-b-poly(NIPA-r-R6GMED) in D2O at pH 7.4 which were recorded with same number of scans and increasing solution temperature. (b) DLS size distribution profile of the same polymer at two representative pH (7.4 and 4.2) and two different temperatures.

homopolymer (33 °C). As a result, above 25 °C (CAT) the poly(NIPA-r-R6GMED) block turns hydrophobic and undergoes dehydration to form aggregates from hydrated unimers. In order to further ascertain the temperature induced transition from unimers to aggregates of poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer, DLS study was effectively used by us. Figure 4b shows the DLS data of the copolymer solution at temperatures below (15 °C) and above (37 °C) the CAT as well as at two different solution pH of 7.4 and 4.2. At both the pH values, the copolymer was seen to exist as hydrated unimers having an average hydrodynamic diameter ( ) varying from ~ 8 to10 nm at 15 °C. At 37 °C i.e. above the CAT, the unimers self-

assembled to form nanostructures with diameter ( ) of ∼160 nm at pH 7.4 which was slightly


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increased to ~180 nm on lowering the pH of the solution to 4.2. The correlograms of the DLS analysis are provided in Figure S13 (Supporting Information). This slight increase in diameter is attributed to the slight decrease of hydrophobicity R6GMED units due to transformation of spirolactam to ring-opened amide form at lower pH. TEM study of the copolymer solutions were performed at 37 °C at both the pH values for better understanding of the nature of these self-assembled nanostructures and the effect of pH on the self-assembly phenomenon. Studies done at pH 7.4 and 4.2 suggested the formation of nanosized spherical aggregates having diameter ∼200 ± 50 nm. A darker thin wall and a hollow interior indicated the formation of vesicular assembly (Figure 5a and b). The particle sizes obtained from TEM (Figure 5a, b) were slightly higher than those observed from the DLS studies. This could be attributed to flattening of the vesicles onto the TEM grid at the time of adsorption during sample preparation. A similar observation has been reported earlier as well.42 In order to preserve the actual structure in the solution, we have performed cryo fixation during TEM measurement. Cryo-TEM images show clear bilayer which further ascertains the formation of vesicle from the block copolymer in both the solution pH 7.4 and 4.2. The cyro-TEM images are presented in Figure 5c and 5d for the two pH values. In this case, the sizes obtained were closely matched with the sizes obtained from DLS measurement. The histograms of the size distribution have been provided in the Supporting Information (Figure S14).


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Figure 5: TEM images of the vesicles obtained from 0.88 mM aqueous poly(PEGMA)-bpoly(NIPA-r-R6GMED) copolymer solution at (a) pH 7.4 and (b) pH 4.2 by maintaining the solution temperature of 37 °C in both the cases. (c) and (d) shows the cryo-TEM images of the vesicles at pH 7.4 and 4.2 respectively corresponding to the above-mentioned copolymer solution in similar condition.

Hydrophilic Dye Encapsulation and Release: We have further ascertained the temperatureinduced spontaneous formation of vesicle by encapsulation of hydrophilic guest molecule and its subsequent release. To investigate this, hydrophilic Rhodamine B (RB) dye was encapsulated into the nanostructures by means of sonication of an aqueous solution of the poly(PEGMA)-bpoly(NIPA-r-R6GMED) copolymer at physiologically relevant condition of 37 °C and pH 7.4. Extensive dialysis was carried out subsequently to enable complete removal of the free or nonencapsulated RB molecules. Separately, another RB solution was prepared in phosphate


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buffer (without any polymer, at 37 °C, pH 7.4) having same absorbance value as the dialyzed solution. Emission spectra of the two solutions having same absorbance were recorded and presented in Figure 6. RB solution showed emission maxima at 584 and 581 nm in the absence and presence of copolymer vesicle respectively. The fluorescence emission intensity at 581 nm of the RB molecules encapsulated into the vesicles was significantly lower (∼56%) than the emission intensity obtained at 584 nm from an absorbance matched polymer-free aqueous RB solution under similar condition. The resulted attenuation of the fluorescence intensity is due to the self-quenching resulting from the confinement of RB molecules inside the aqueous interior of the vesicles which further establishes the formation of vesicular assemblies. Moreover, we have also determined the quantum yield of RB in vesicular solution with and without dialysis at pH 7.4 and 37°C, and the values were 0.17 and 0.37 respectively. The quantum yield of RB in water was previously reported to be 0.31 under physiological condition.43 Therefore, the decrease of fluorescence quantum yield is certainly due to the encapsulation of RB molecules into the aqueous core of the vesicles and subsequent self-quenching. This self-quenching nature of the dye molecules resulting from confinement has been utilized earlier also for ascertaining the formation of vesicles.21,44-45 Additionally, when the temperature of the RB encapsulated vesicles (obtained after dialysis at 37 °C as described earlier) was lowered to 15 °C (to break the vesicles) and fluorescence intensity was measured without any further dialysis. The emission intensity was increased by ∼1.5 times (Figure 6) and reached close to that obtained for the polymer-free RB solution. The above results undoubtedly establishes the disruption of the vesicular structure on decreasing the solution temperature from 37 °C to 15°C and thereby releasing the encapsulated hydrophilic guest molecules (RB) from the core of the vesicles to bulk water.


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Figure 6: Absorbance-normalized fluorescence spectra of hydrophilic dye RB encapsulated in 0.88 mM poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer vesicle at pH 7.4 and 37 °C and then temperature-induced release of encapsulated RB from vesicle.

Investigation of FRET Process by Steady-State and Time Resolved Fluorescence Studies: We have investigated the FRET process from the hydrophobic fluorescent dye, C-153 (as a donor) to polymer-bound R6GMED units (as an acceptor), both located in the bilayer region of vesicles formed by the self-assembly of poly(PEGMA)-b-poly(NIPA-r-R6GMED). Figure 7a shows the steady-state fluorescence emission spectra of C-153 encapsulated in the vesicles at pH 7.4 at 37 °C. According to a previous report, C-153 shows very weak fluorescence and the emission maxima lies at 550 nm with a quantum yield of 0.12 in water. 32-33, 46 On encapsulation of C-153 in the vesicles formed by the block copolymer, the emission maxima was blue-shifted to 502 nm and the quantum yield was significantly increased to 0.43. Figure 7a also shows the UV-vis absorption spectra of only block copolymer vesicular solution (in absence of C-153) with an absorbance maxima at 534 nm at pH 5.0 and at 37 °C. The observed absorbance was due to the ring opened amide form of polymer-bound R6GMED units in one of the blocks. It is noteworthy to mention here that the absorbance of the only block copolymer vesicular solution


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(in absence of C-153) was nearly zero at pH 7.4 and at 37 °C (Figure 7b). This is due to presence of polymer-bound R6GMED units in spirolactum ring form at pH 7.4. Thus, at pH 5.0, existence of significant spectral overlap between donor (C-153) emission and acceptor (R6GMED) absorption (Figure 7a) made the system suitable scaffold for efficient FRET process which was otherwise not possible in pure water/buffer (vesicle free) and in pH 7.4 (vesicular solution).

Figure 7: (a) Overlap between emission spectra of C-153 confined in the vesicles at pH 7.4 and absorption spectra of poly(PEGMA)-b-poly(NIPA-r-R6GMED) solution in absence of C-153 at pH of 5.0 and at 37 °C. (b) Absorbance of only block copolymer at two different solution pH 7.4 and 5 at 37 °C.

The FRET process of encapsulated C-153 was also studied at varying solution pH of poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer solution at 37 °C using steady-state and time resolved fluorescence measurement. Concomitant decrease of C-153 (donor) emission intensity with increase in the acceptor fluorescence intensity indicates occurrence of effective FRET (Figure 8a). The decrease in the lifetime value of vesicle encapsulated C-153 on lowering the solution pH from 7.4 to 5.0 and 4.2 clearly confirmed the occurrence of effective FRET (Figure 8b and Table 2). At pH 7.4, the average lifetime of the encapsulated C-153 in the vesicle shows a value of 4.09 ns whereas on changing the solution pH to 5.0 and 4.2 the average lifetime


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value decreases to 2.33 ns and 1.84 ns respectively. The values of each of the lifetime components of bi-exponential fluorescence decay of vesicle encapsulated C-153 were also decreased (Table 2). We have also determined lifetime of C-153 in phosphate buffer (PB) without vesicles at pH 7.4 and 4.2. No significant difference in the lifetime values between the two pH was observed (Table 2 and Figure S15a), which further supports FRET phenomena in presence of vesicle.

Figure 8: (a) Steady-state fluorescence spectra of encapsulated C-153 at 37 °C as a function of different solution pH in poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer solution. (b) Time-resolved fluorescence decay of donor C-153 with varying solution pH in the aqueous polymer solutions corresponding to the above-mentioned condition.

Table 2: Decay parameters of C-153 with varying solution pH in presence and absence of 0.88 mM poly(PEGMA)-b-poly(NIPA-r-R6GMED) copolymer solution at 37 °C. System C-153 in vesicle C-153 in vesicle C-153 in vesicle C-153 without vesicle C-153 without vesicle #

Solution pH /0 (ns) 7.4 5.0 4.2 7.4 4.2

1.70 0.95 0.82 1.62 1.73

12

0.54 0.49 0.63 1.00 1.00

/3 (ns)

Error is ±10% in all TCSPC result.


6.90 3.66 3.59 -

1)

0.46 0.51 0.37 -

〈/〉 (ns) 4.09 2.33 1.84 1.62 1.73

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FRET parameters were calculated using the Förster theory, in which the rate expression for fluorescence resonance energy transfer is given by the following equation, 678 =

2

; 9:

=

( >;)@ where

r is the distance between centers of the donor and acceptor molecules, R0 is the Förster distance i.e. the distance at which the energy transfer efficiency is assumed to be 50%, AB is the lifetime of the donor in absence of the acceptor. R0 can be calculated using the following equation, C@ =

D(EF 2)GH Ф: S O PB (,)QR (,) ,T U, 2)IПK LMN

where n is the refractive index of the medium, V ) is the

orientation factor of the two interacting dipoles, N is the Avogadro’s number, ФB is the fluorescence quantum yield of the donor in the absence of the acceptor, PB (,) is the normalized S

fluorescence intensity of the donor in absence of acceptor which implies O PB (,)U, = 1, and

QR (,) is the molar absorption coefficient of the acceptor generally represented in mol cm unit. -1

-1

The calculated spectral parameters are listed in Table 2 and Table 3. The equation 〈A〉 = A2 X2 +

A) X) , where A2 and A) are the lifetime components with relative weights of X2 and X)

respectively, was used to analyze the bi-exponential decay. In this work, the energy transfer efficiency was calculated from the average lifetime quenching data. As mentioned earlier that in the present case, both the donor and acceptor molecules were located completely inside the hydrophobic bilayer. The observed change in the FRET efficiency with solution pH was due to reversible transformation of spirolactam structure to ring-opened amide form of R6GMED units present in one of the block segments in the copolymer, which act as an acceptor in these FRET process (Figure S15b, Supporting Information). With decreasing solution pH, more number of transformation of spirolactam structure to ring opened amide form of R6GMED unit occurs thereby increasing the probability of locating a suitable energy acceptor in the proximity of the donor (C153).


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Table 3: FRET parameters for C-153 and R6GMED pair located in polymer vesicles. Solution pH ,- (nm) Z J(,) (M-1 cm-1 nm4) C (Å) E (%) # r (Å) #

#

678 (s-1) #

7.4 5.0

408 408

0.43 0.43

7.90 × 1012

18.7

43

19.6

1.85 × 108

4.2

408

0.43

9.13 × 1012

19.1

55

18.4

3.06 × 108

From time-resolved measurement.

Table 3 provides the acceptor fluorescence quantum yield dependence of FRET rate and efficiency with a fixed donor concentration. Furthermore, it is obvious that on lowering the solution temperature below CAT at a particular pH (acidic medium, pH ≤ 6), the quantum yield of the encapsulated C-153 (donor) should be decreased due to disassembly of the vesicles thereby eliminating the energy transfer efficiency. The change in quantum yield of the fluorophore plays a dominant role since in a system like this, the rate and efficiency of energy transfer from donor to acceptor is governed by stimuli like temperature and pH as a result of altered quantum yield of the fluorophores. It can be inferred from the above result and discussion that the present poly(PEGMA)-b-poly(NIPA-r-R6GMED) vesicular system are able to provide a suitable scaffold for occurrence of the FRET process and this type of FRET-based stimuliresponsive block copolymer nanocarriers with tailored morphology have great potential in realtime monitoring of the release of the core-encapsulated probes/drugs into the biological media. This is true even if the probe/drug molecules are non-fluorescent.

CONCLUSION In conclusion, a poly(ethylene glycol) containing thermally responsive and pH switchable fluorescent diblock copolymer poly(PEGMA)-b-poly(NIPA-r-R6GMED) was successfully


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synthesized by RAFT polymerization which self-assembled to form nanostructured vesicle above CAT (~25 °C) in aqueous solution under physiologically relevant condition. This vesicle is capable of encapsulating and release both hydrophobic and hydrophilic molecules. This thermoresponsive vesicle undergoes changes in pH dependent fluorescence property due to reversible transformation of the spirolactam structure to ring-opened amide form of R6GMED units, which are randomly present along with NIPA units in one of the block segments in the copolymer. Moreover, it has also been demonstrated that the present poly(PEGMA)-bpoly(NIPA-r-R6GMED) vesicular nanocarriers can act as a scaffold enabling highly efficient FRET process between encapsulated C-153 (donor) and R6GMED units (acceptor) in acidic pH relevant to the cytoplasm and the cell nucleus in leukemic tissues. Therefore, this FRET-based stimuli-responsive vesicle will be of general interest as a biocompatible carrier of various bioactive entities for the imaging and/or treatment of cancers, even if the drug is nonfluorescent, by monitoring effective intraoperative change in FRET parameters inside the leukemic cells and hence, may lead to optimal clinical outcomes in the success of cancer chemotherapy.

ACKNOWLEDGMENT Financial support from Science and Engineering Research Board, Department of Science and Technology, Government of India (Project Ref No: EMR/2016/007040) is acknowledged. Authors also thank Indian Institute of Technology Kharagpur for funding the purchase of a DLSZeta and a multi-detector GPC instrument through competitive research infrastructure seed grants (project codes ADA, NPA with institute approval numbers - IIT/SRIC/CHY/ADA/201415/18 and IIT/SRIC/CHY/NPA/2014-15/81 respectively). Authors thank Prof. N. Sarkar for


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providing facility for fluorescence lifetime measurements. C. Maiti acknowledges UGC, Govt. of India, New Delhi for research fellowship. SUPPORTING INFORMATION Detail synthesis procedures for N-(Rhodamine-6G)lactam-N'-methacryloyl

ethylenediamine

(R6GMED) and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPPA), 1HNMR and

13

C

NMR spectra of some essential compounds, GPC chromatograms of the polymers, plot of fluorescence intensity of Nile Red, some more DLS and fluorescence data are provided in the Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected], [email protected] Ph no: +91-3222-282326; Fax: +91-3222-282252 Orchid ID: 0000-0003-2574-5378

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Energy-Transfer Phenomena in Thermoresponsive and pH

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