Fluorescence Resonance Energy Transfer (FRET): A Powerful Tool for

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Review Article

Fluorescence Resonance Energy Transfer (FRET): A Powerful Tool for Probing Amphiphilic Polymer Aggregates and Supramolecular Polymers Priya Rajdev, and Suhrit Ghosh J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b09441 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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The Journal of Physical Chemistry

Fluorescence Resonance Energy Transfer (FRET): A Powerful Tool for Probing Amphiphilic Polymer Aggregates and Supramolecular Polymers Priya Rajdev *a and Suhrit Ghosh * a, b

a

Technical Research Center; b School of Applied and Interdisciplinary Sciences; Indian Association for

the Cultivation of Science, 2A and 2B Raja S. C. Mullick Road, Kolkata, India- 700032

Email: [email protected]; [email protected]

ABSTRACT This review article highlights the utility of the Fluorescence Resonance Energy Transfer (FRET) to probe the dynamics and related issues in amphiphilic polymeric aggregates and supramolecular polymers. Amphiphilic polymers are attractive over their small molecule analogues because they exhibit significantly lower critical aggregation concentration, relatively larger particle size (suitable for the Enhanced Permeation and Retention Effect) and a much slower dynamics of exchange between the unimer and aggregate. Representative examples on exchanges dynamics in amphiphilic polymer aggregates and their non-covalent encapsulation stability as a function of the structure of the macromolecule, cross-linking, environmental parameters and biological conditions, as probed by FRET studies, have been included in this article. Further a related discussion on the utility of FRET in studying the exchange dynamics in supramolecular polymers, particularly in aqueous medium, has been discussed in length which reveals a strong impact of chirality, side chain polarity and other parameters. Overall, the article brings out the strength of this technique to probe dynamics 1 ACS Paragon Plus Environment

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of aggregates and assembled systems, mostly in water medium, which has a paramount importance in designing future biomaterials.

INTRODUCTION

Fluorescence Resonance Energy Transfer (FRET)1 is the energy transfer from a fluorescent donor to an acceptor molecule (chromophore/fluorophore) during which the energy from the excited donor (D) chromophore is transferred to the acceptor (A) molecule in the ground state through a radiationless phenomenon without the actual emission of the photon. The acceptor molecule then gets excited and emits to reach its ground state if it is a fluorophore. On the other hand, if the acceptor molecule is non-fluorescent in nature, there is quenching. FRET was first observed by Theodor Förster in the late 1940’s 2 and hence it is also known as Förster Resonance Energy Transfer. The efficiency (E) of the FRET process strongly depends on the distance between D and A chromophores and thus it has emerged as a highly sensitive tool to probe complex phenomenon in various fields of biology3-6 including DNA sequencing and detection, protein folding, protein-protein interaction, in vivo imaging, in vitro assays and so on. Very high sensitivity (to a single molecular level), selectivity, lower measurement timescale, lack of interference by other molecules or solvent, applicability to dilute samples, the effective range of distance and non invasive nature are a few notable advantages offered by the FRET technique. Apart from biological problems, FRET has also been extensively studied in context of sensing, optoelectronic properties including light harvesting and others, assembly / disassembly of stimuli-responsive polymers, which have been described elsewhere

7-10

and fall out of the scope

of this review. This article particularly focuses on the relevance of FRET in context of the 2 ACS Paragon Plus Environment

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dynamics of amphiphilic polymeric aggregates and supramolecular polymers and related events. Aggregation of amphiphilic polymers in block selective solvents leads to various elegant nanostructures11 and is a topic of current interest due to the potential application12 in drug and gene delivery, biosensing, development of antibacterial materials, nanoelectronics and so on. They are more useful over various small molecule surfactants because they exhibit significantly lower critical aggregation concentration, suitable particle size to get the advantage of the Enhanced Permeation and Retention (EPR) effect13 and a much slower dynamics of exchange between the unimer and aggregate. Such kinetic stability is particularly important for biological applications because if the dynamics is slower, the probability of premature release of entrapped drug molecules can be reduced which is essential to minimize any undesired side effect. Hence, it becomes of utmost importance to probe the stability/ dynamics of such amphiphilic polymeric aggregates to judge their potential for biological applications. While multiple techniques (spectroscopy, microscopy and light scattering) have been used to gather information14 on the morphology, size, shape and guest encapsulation ability of the amphiphilic polymeric aggregates, it is rather challenging to probe their dynamics. In this context, FRET has been successfully utilized to monitor such events in real time and also in very dilute solutions. Therefore, the pros and cons of this technique for studying the dynamics of amphiphilic polymer aggregates is indeed important in context of the design and function of amphiphilic polymers, which is precisely the focus of this article. Further we have included a related discussion on the utility of FRET in studying dynamics of supramolecular polymers15 which are emerging as a very important class of materials with relevance in biology and optoelectronics. Dynamics is inherent in supramolecular polymers which makes them adaptable, self-healing in nature and endows other unique properties. This has been probed by FRET and the article will collate those limited

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recent examples to highlight the importance of understanding the dynamics of supramolecular polymers for their functional utility.

DISCUSSION

FRET Principle: The theory of FRET 1-2, 16 involves a radiationless energy transfer from D to A via dipolar coupling when there is a resonance of the donor emission and the acceptor excitation dipoles, which also justifies the term Resonance Energy Transfer. The energy transfer process is schematically described by the Jablonski diagram shown in Figure 1a. The efficiency (E) of this process can be estimated either from equation 1 or 2.

E = 1- (IDA/ID)

(1)

E = 1 – (τDA/ τD)

(2)

ID and IDA are the intensities of the donor emission in the absence and presence of the acceptor, respectively. τDA and τD are the fluorescence lifetime of the donor in presence and absence of the acceptor, respectively. The above equations are valid for a single pair of D and A with a fixed distance r. E varies with r as per equation 3.

E = R06/(R06+ r6)

(3)

Where R0 is the Forster distance, i.e. the distance at which E = 50 % and can be determined using equation 4.

R0 = 0.211(κ2n-4QD J(λ) )1/6 (in Å)

(4) 4 ACS Paragon Plus Environment

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Where, κ2 is the orientation factor which ranges from 0 (for perpendicularly oriented dipoles) to 4 (dipoles parallel to each other). For a random and dynamic distribution of the fluorophores, it has a fixed value of 2/3. n is the refractive index of the medium and for aqueous solution, it has a value of 1.33. QD is the fluorescence quantum yield of the donor. Overlap integral J(λ) measures the extent of the spectral overlap (Figure 1b) between the normalized donor emission [FD(λ)] and the acceptor absorption spectra (εA) and is determined by the equation 5.

(5)

Figure 1: (a) Jablonski diagram for describing FRET from donor (D) to acceptor (A); (b) Spectral overlap between the D (DiO) emission (dashed, blue) and A (DiI) absorption (solid, red) for FRET to occur. Upon the excitation of D, the energy is non-radiatively transferred to A, which is at a distance r from the D. The shaded region represents the overlap region between the donor emission and the acceptor absorption spectra.



Scheme 1: Examples of a few commonly used FRET D-A pairs such as naphthalene(D1)pyrene(A1), DiO(D2)-DiI(A2), FITC(D3)-Rhodamine(A3), Anthracene(D4)-BODIPY(A4) and Cy3 (D5)-Cy5(A5) utilized in polymer and supramolecular systems. Donor and acceptor dyes have been shown in blue and red, respectively.

Hence, the rate of energy transfer (kT) depends on (i) the extent of the spectral overlap, i.e. J(λ), greater the overlap, higher is the efficiency (ii) the quantum yield of the donor (iii) the separation distance r between the donor and acceptor molecules and (iv) the relative orientation of the donor emission and the acceptor excitation dipoles. Over the years, several FRET D-A pairs have been used depending on their photophysical properties and feasibility to chemically attach them with 6 ACS Paragon Plus Environment

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the system(s) under investigation using facile synthetic methodology. Among them, Naphthalene/Pyrene, Naphthalene/Coumarin, FITC (Fluorescein isothiocyanate)/Rhodamine, DiO

(3,3′-

Dioctadecyloxacarbocyanine)

/DiI

(1,1′-dioctadecyl-3,3,3′,3′-

tetramethylindocarbocyanine perchlorate) are a few commonly used FRET pairs (Scheme 1) which have been frequently used for studying the dynamics of polymer/ supramolecular assemblies..

Exchange dynamics in amphiphilic polymer aggregates by FRET: Amphiphilic polymers produce wide ranging aggregated structures in water 11-12, 14 which remain in equilibrium with the unimer. Therefore the overall dynamics of a particular system may be realized by estimating the exchange dynamics of a unimer between the aggregates. For small molecule surfactants, such dynamics of exchange is known to be very fast (in the time scale of millisecond to submillisecond)17 while

it

is known to be rather slow for the amphiphilic polymeric

aggregates.18 The lifetime of diblock copolymers in their micellar aggregate has been reported to be few hours or even longer.19-22 Although experimental methods such as sedimentation velocity or size exclusion chromatography were demonstrated 17-19 to study such exchange dynamics of amphiphilic polymer aggregates, FRET was soon recognized as an attractive spectroscopy tool to study such process.20-22 It operates based on a simple principle as depicted in Scheme 2. Basically, a given amphiphilic polymer can be labeled (preferably in the junction or in the hydrophobic domain) independently with a FRET donor and a FRET acceptor and then their individual micelles are physically mixed. If the aggregate is fully frozen, there should not be any cross-talk between the D and A chromophore and thus no FRET appears over time. On the other hand, if there is a dynamic exchange, gradually all the aggregates will be populated by D and A labeled polymers and thus FRET will evolve gradually depending on the rate of the exchange. 7 ACS Paragon Plus Environment


Scheme 2: Schematic describing the working principle of FRET for probing exchange dynamics of amphiphilic polymeric aggregates. Aggregates prepared separately by FRET donor (shown by green dots) or acceptor (shown by red dots) labeled polymers, when physically mixed, they hybridize to produce mixed aggregates with close proximity of the D and A dyes for FRET to emerge. In one such early example,

22

Mattice and coworkers studied FRET in polystyrene-block-

poly(oxyethylene), labeled with either naphthalene or pyrene in the junction of the hydrophobic and hydrophilic block. A mixture of the pre-formed aggregates of these two separately labeled polymers showed strong FRET after equilibration for sufficient duration (Figure 2), suggesting co-localization of the D and A labeled polymers in the same micelle due to the dynamic exchange of the polymers between micelles. Time dependent variation of the donor emission intensity (Figure 2) revealed initial fast decrease followed by a slow decrease which could be fitted to a sum of two exponentials. The difference in time constants varied by more than an order of magnitude which could not be rationalized only by the model of chain exchange through unimer. Therefore an additional process such as chain exchange of chains through micellar collision was predicted in this report. In a relatively recent report,23 co-existence of such multiple exchange mechanism has been described elaborately in aggregation of naphthalene/ pyrene labeled poly(ethylene oxide)-block-poly(methacrylic acid) by self-complexation. The time 8 ACS Paragon Plus Environment

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constants were determined using equation 6, where f1 , k1 and f2, k2 are parameters for the fast and slow events, respectively. (6)

Figure 2: Left- Emission spectra (excitation of naphthalene chromophore) of (a) naphthalenelabeled polystyrene-block-poly(oxyethylene) in methanol and (b) methanol containing pyrenelabeled copolymer; Right-Normalized fluorescent emission intensity at 338 nm in 90:10 MeOH/ H2O from a mixture of the naphthalene- and pyrene-labeled copolymers as a function of time at 25, 30, 35 and 40 °C from top to bottom. Reprinted with permission from ref. 22. Copyright 1995 American Chemical Society. At low pH, they remained in inter-molecularly complexed states. Exchange of chains between such aggregates was probed by the FRET which revealed comparable contribution from (i) insertion and expulsion of single chains and (ii) merging and splitting of the micelles in the overall exchange process (Figure 3). It was observed that with increase in the thickness of the corona (by increasing the block length), the overall exchange dynamics became slower and the pathway (ii) dominated over the pathway (i).



Figure 3: Two separate chain exchange mechanisms in self-complexed aggregates of poly(ethylene oxide)-block-poly(methacrylic acid). Reprinted with permission from ref. 23. Copyright 2004 American Chemical Society. .

Figure 4: Top- Structure of D- and A- labeled amphiphilic block copolymers having hydroxyl groups in the hydrophobic block; Bottom- Schematic showing remarkably stable mixed micelle


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by supramolecular cross-linking in the core as probed by FRET studies. Adopted and reprinted with permission from ref. 24. Copyright 2015 American Chemical Society. We have recently reported

24

the impact of supramolecular networking in the core of an

amphiphilic diblock-copolymer micelle on its exchange dynamics (Figure 4). PEO-b-PMMA-coPHEMA amphiphilic copolymer was separately labeled with a green and red emitting FRET DA pair. These polymers individually formed micellar aggregates in water. When preformed micelles of the red and green labeled polymers were mixed, the FRET intensity gradually increased and took a rather long time (>50 h) to saturate, indicating an unusually slow exchange rate. When the OH groups were blocked by an acetyl group, the polymers exhibited much faster dynamics as the FRET ratio reached the maximum values by only ~ 3 h. Similarly in presence of TFA (that protonates the OH groups and thereby destroys the H-bonding), the mixing was completed within ~ 2h. These results strongly evoked that the free OH groups indeed made a strong impact to provide the stability of the micellar aggregates by cross-linking the core through H-bonding which was also supported by FT-IR studies. FRET based strategies to study exchange dynamics have also been employed for protein-functionalized amphiphiles25 (Figure 5).

Figure 5: Top- Schematic showing evolution of FRET as a consequence of exchange of chains between aggregates of protein-functionalized amphiphiles; Bottom- FRET based method for 11 ACS Paragon Plus Environment


determination of average aggregation number. Reprinted with permission from ref. 25. Copyright 2010 American Chemical Society. Two fluorescent proteins namely ECFP and EYFP (FRET pair) were linked with a micelle or liposome, derived from pegylated phospholipids, by the Native Chemical Ligation. In sterically stabilized protein functionalized liposomes, no FRET appeared after mixing the ECFP and EYFP functionalized pre-formed aggregates, indicating a frozen structure without any significant exchange. In contrast, the protein functionalized micellar aggregates were found to be highly dynamic showing t1/2 of exchange ~ 2h at rt which reduced to a few minutes at 37 °C. The study not only unfolded such sharp contrast in the chain exchange dynamics of liposome and micellar aggregates, but also demonstrated the utility of FRET to estimate the aggregation number of such micellar aggregates. Co-assembled micelles containing both ECFP and EYFP were prepared which expectedly showed a strong FRET. It was gradually titrated with a non-labeled micelle. Due to the dynamic exchange, the protein labeled lipids were distributed among all the micelles resulting in gradual decrease in the FRET intensity which eventually diminished. This was assumed to be the situation when none of the micelles contained more than one protein-attached lipid. Therefore, from this experiment and the relative concentration of the two micelles at the end of the titration, it was possible to estimate the average aggregation number. In fact they also could estimate the critical micellar concentration from the FRET experiment.

Amphiphilic aggregates with non-covalently encapsulated D/A chromophores and FRET studies: The forgone discussion described representative examples of exchange dynamics of chains in amphiphilic aggregates using covalently attached D/A FRET pairs. Such amphiphilic aggregates have been used for non-covalent guest encapsulation as that mimics their potency as


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drug delivery vehicle. Faster exchange of the polymer chains between aggregates makes a leaky container which is not desirable for the target specific drug delivery application. However, a more direct estimate of the non-covalent encapsulation stability of such containers may not solely depend on the exchange dynamics of the chains but it may also be related to other associated parameters such as the temperature, pH, physical and chemical properties of the guest and so on. Therefore FRET studies with non-covalently D and A co-encapsulated aggregates have emerged as an attractive strategy for estimating the intrinsic non-covalent encapsulation stability as well as the stability of the container in presence of biologically relevant external triggers. Thayumanavan and coworkers have extensively studied non-covalent encapsulation stability of polymeric nanogels (NG). An amphiphilic statistical copolymer with appended pyridyl-disulfide (PDS) group was used to prepare the nanogel (NG) in presence of a hydrophobic guest (DiO or DiI) which are well known FRET D-A pair (Figure 6).26

Figure 6: Schematic showing non-covalent encapsulation stability of redox-responsive nano-gels as probed by FRET. Reprinted with permission from ref. 26.

Copyright 2010 American

Chemical Society. A redox reagent Dithiothreitol (DTT) was used for the synthesis of the NG. DTT reduced a few of the pendant PDS groups to produce the free thiol which in turn reacted with the rest of the PDS groups to produce the disulfide cross-linked NG, encapsulated with either DiO and DiI. By varying the amount of DTT, the cross-link density could be tuned. When DiO and DiI entrapped NGs were mixed together, the DiO emission reduced gradually with concomitant increase in the 13 ACS Paragon Plus Environment


DiI emission due to FRET. It was attributed to the co-localization of the two guests in the core of the NG as a result of the dynamic exchange. Soon after mixing, an increase in the FRET ratio [IA/(IA +ID)] as was noticed which showed a linear relationship with time. The slope was termed as the leakage coefficient (Λ, h-1). It was found to be remarkably slow for the NGs compared to other aggregates such as CTAB micelle, structurally similar non cross-linked random copolymer aggregate or commercially used Pluronic block copolymer micelle. It was shown that with increase in the cross-link density, Λ could be reduced further. Interestingly DTT, the same reagent that was used for cross-linking, when used in excess, de-cross-linked the NG by reducing the disulfide bridge and released the entrapped guest molecules which could also be probed by FRET. Subsequently, they examined other NGs with different size, explored drug delivery27 and probed influence of Hofmeister ions on the non-covalent encapsulation stability by FRET.28 They also elucidated

29

the guest-exchange mechanism in such NGs (Figure 7). Similar to what

has been discussed in the previous section on the chain exchange mechanism, guest exchange pathways in the NGs were found to be: (i) diffusion controlled or (ii) host-collision controlled phenomenon. By extensive FRET experiments, it was concluded that the pathway (ii) was dominant when the core was sufficiently hydrophobic, while it shifted to pathway (i) with decreasing hydrophobicity of the core which could be tuned in this case by pH.

Figure 7: Two possible pathways for guest-exchange between separate aggregates as elucidated by environment dependent FRET studies. Reprinted with permission from ref. 29. Copyright


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2014 American Chemical Society. Further permissions related to the material excerpted should be directed to the ACS.

We have tested30 encapsulation stability in amphiphilic random copolymer aggregates by FRET. A series of amphiphilic random copolymers (Figure 8) were prepared by post-polymerization modification which differed only in the length of the hydrophilic pendant chain. All of these polymers showed aggregation in water with very low CAC. Non-covalent encapsulation stability was probed by monitoring the FRET with time after mixing the DiO and DiI encapsulated micelles in similar fashion that has been reported earlier by Thayumanavan and coworkers.

Figure 8: Top-Structure of amphiphilic random copolymers; Bottom-Emission spectra of the physically mixed aggregates of P-3OE, separately encapsulated with DiO and DiI FRET D-A dyes. No significant FRET evolved even after 100h indicating stable aggregates. Adapted and Reprinted from ref. 30. Copyright 2013 John Wiley and Sons. We found that these simple un-cross-linked random copolymer aggregates provided an enormous non-covalent encapsulation stability as no significant FRET appeared even at 100 h after mixing. 15 ACS Paragon Plus Environment


The leakage coefficients in these systems were found to be very low. It was postulated to be a consequence of H-bonding network formation in the confined hydrophobic domain of the aggregates involving the backbone amide functionality with the ester and/or the ether oxygen atoms, present at the vicinity in the aggregates. Such exchange dynamics between encapsulated guest molecules in polymer micelle has also been studied31 in the intra-cellular environment by Raymo and coworkers. They have used amphiphilic polymethacrylate backbone with pendant decyl and oligo (ethylene glycol) chains which formed nanoparticle and could sequester hydrophobic guests such as anthracene (FRET donor) and BODIPY (FRET acceptor). Physical mixing of the anthracene and BODIPY entrapped nanoparticles resulted in a rapid evolution of FRET suggesting a fast exchange. These co-encapsulated nanoparticles showed efficient cellular internalization and the intra-cellular FRET confirmed an intact container inside the cell. Interestingly even when the cells were incubated sequentially with anthracene-loaded nanoparticles followed by BODIPY-loaded nanoparticles, the FRET appeared quickly (Figure 9) inside the cell suggesting such dynamic exchange could also operate in the intra-cellular environment.

Figure 9: Fluorescent images of HeLa cells after consecutively treated with donor and acceptorentrapped polymer aggregates in two possible sequences (a- D followed by A, b-A followed by


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D). In both occasions images were captured after 3 h of incubation. Note that the green emission appears from the acceptor chromophore while in both cases the excitation was for the donor chromophore indicating it to be FRET as a consequence of dynamic exchange even in the intracellular environment. Reprinted with permission from ref. 31.


Chemical Society.

While these examples demonstrate intrinsic dynamics of the chain/ guest exchange, FRET has also emerged as an excellent tool for probing externally triggered (dilution, solvent, pH, serum, cellular environment etc.) disassembly of amphiphilic polymer aggregates.32-35 For this purpose, unlike the previous examples, both of the D and A chromophores are encapsulated simultaneously in a single aggregate resulting in strong FRET due to the close proximity of the two dyes in the confined core of the aggregate. Thus diminishing FRET emission could be used to check the disassembly. In the recent past we have reported32 the self-assembly of a molecularly engineered hydrophilic polymer (Figure 10) to highly stable polymersome structure due to hydrophobically assisted H-bonding.



Figure 10: Top- Structure of molecularly engineered hydrophilic polymer; Bottom- Emission spectra (excitation at donor chromophore) of DiO (D) and DiI (A) co-encapsulated aggregates as a function of good solvent. Diminishing acceptor emission with concomitant regain in the donor emission with THF addition indicates disassembly. Adapted and reprinted with permission from ref. 32. Copyright 2013 American Chemical Society.

To examine the effect of a good solvent on the stability we co-encapsulated DiO and DiI FRET pair in their polymersome structure which resulted in strong FRET. Sensitivity of this otherwise highly robust assembly was examined in presence of a good solvent THF. The FRET almost disappeared with only 30 % THF, suggesting high sensitivity of the structure to a “good” solvent. Probing such disassembly of polymer aggregates in vivo is relevant for cargo delivery which has been demonstrated by Cheng and co-workers using FRET.33 DiOC18-DiIC18 FRET pair was encapsulated in the hydrophobic core of poly(ethylene glycol)-poly(d,l-lactic acid) (PEG-PDLLA) micelle (Figure 11).

Figure 11: Schematic showing co-localized DiOC18-DiIC18 FRET dyes (indicated by gree and red dots) in PEG-PDLLA micelle escape as a consequence of globulin induced disassembly as probed by FRET. Reprinted with permission from ref. 33. Copyright 2008 American Chemical Society. As expected the dual dye loaded micelle exhibited strong FRET. When it was administered to an animal by intravenous injection, the FRET efficiency reduced significantly after only 15 min, 18 ACS Paragon Plus Environment

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indicating the release of the entrapped D and/or A chromophores quickly in vivo. FRET studies further indicated dominant role of the α- and β-globulins for the observed fast release, while γglobulins, albumin, and red blood cells played a minor role. Shoichet and coworkers investigated34 the stability of poly (D, L-lactide-co-2-methyl-2-carboxytrimethylene carbonate)g-poly (ethylene glycol) aggregate (Figure 12) in serum and in presence of individual serum protein solutions by FRET. In this case also DiO-DiI FRET pair was co-encapsulated in the micellar container producing an efficient FRET. In presence of the serum, no significant change was noticed in the FRET ratio over long duration, indicating their excellent in vitro stability. The contrasting results in these two studies highlight the importance of in vivo experiments to estimate the true potential of a drug delivery system for practical application.

Figure 12: Structure of the highly serum-stable poly (D, L-lactide-co-2-methyl-2carboxytrimethylene carbonate)-g-poly (ethylene glycol) polymer aggregate co-encapsulated with FRET D-A pair. Reprinted with permission from ref. 34.


Chemical Society. Further permissions related to the material excerpted should be directed to the ACS.

FRET has also been explored to probe cellular uptake and intra-cellular delivery of encapsulated guest molecules in cell. Many examples have shown non-covalent encapsulation of drug



molecules in polymeric micelles and intra-cellular delivery. Do the entrapped drugs remain inside the micelle till the delivery or they escaped out of the container before it is up-taken by the cell? Such questions were possible to address by elegant FRET experiments. Chen, Li, Wang, Park and Cheng reported

35

a comprehensive study on the intra-cellular delivery process of

encapsulated cargo in a monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) micelle by FRET. The polymer was labeled with a green fluorescent dye (FITC) while a red fluorescent dye DiI was non-covalently entrapped in the core of the micelle. By fluorescence imaging it was observed (Figure 13) that the entrapped DiI entered the cell much before than the polymer indicating escape of the dye in extra-cellular domain from the micellar core. The process was monitored in further detail by FRET-imaging and spectroscopy. For this purpose, DiIC18 and DiOC18 (FRET pair) were co-encapsulated in the micelle and it was incubated with a model vesicle. These studies revealed the delivery of the entrapped dyes in the membrane which acted as a temporary host to facilitate further intra-cellular delivery.

Figure 13: Confocal Fluorescence image of KB cells incubated with FRET micelles containing DiO and DiI. After 2h incubation (A) at 37 ° C and (C) at 4° C. Spectra recorded from a fluorescent area inside of cells and extracelluar space after 2 h incubation of FRET micelles (B) 20 ACS Paragon Plus Environment

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at 37 °C and (D) at 4 ° C. Reprinted with permission from ref. 35. Copyright 2008 National Academy of Sciences.

While these examples demonstrate a complete disassembly of aggregates/release of aggregated dyes in presence of external trigger, we recognized FRET could even be used as a very senstitive tool to probe pH-responsive swelling-deswelling phenomenon in amphiphilic polymer aggregates as it significantly alters the volume of the core and thus effective concentration of the entrapped D-A molecules (Figure 14).36

Figure 14: Top- Schematic showing pH-responsive reversible swelling of D and A coencapsulated polymer aggregate; Bottom- Impact of swelling and de-swelling on the FRET ratio. Reprinted with permission from ref. 36. Copyright 2013 American Chemical Society.



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An amphibilic diblock copolymer was engineered by partial functionalization of the hydrophobic block with pendant carboxylic acid groups to impart pH responsiveness to its aggregates. It showed a compact micellar aggregate in acidic pH with remarkable stability that was attributed to the H-bonding involving the carboxylic acid groups in the confined hydrophobic domain. However in basic pH, the deprotonation of the carboxylic acid groups resulted in swelling of the particles (evident by DLS, TEM and other studies), but not complete disassembly because the hydrophobic block was only partially functionalized with the acid groups. Two chromophores, namely pyrene (FRET D) and DMN (1,8 dimethoxy naphthalene) (FRET A) were coencapsulated in these aggregates and FRET was monitored as a function of pH. With increasing pH, as swelling happened, the FRET intensity decreased and when the pH was brought back to acidic, it increased again. This suggests with swelling, as the volume of the core expanded, the D-A chromophores were distributed in a larger nano-domain resulting in increase in the average distance between them resultingly in less efficient FRET. O’Reilly and coowrkers studied

37

aggregation of Poly(triethylene glycol acrylate)-b-poly(tert-butyl acrylate) (P(TEGA)-b-P(tBA)) block copolymers, covalently labeled with a dithiomaleimide (DTM) fluorophore either in the hydrophobic or hydrophilic block (Figure 15). These polymers formed micelle in water and exhibited strong fluorecence when the DTM fluorophore was located in the core. In contrast the shell labeled micelles were less emissive due to collisional quenching. Interestingly, the corelabeled micelles could self-report encapsulation of a hydrophobic guest molecule (Nile Red) by onset of FRET between the covalently attached DTM fluorophore and the non-covalently sequestered guest Nile Red.


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Figure 15: Schematic showing core- and shell- labeled micelles and non-covalent encapsulation of complementary fluorophore for FRET studies. Reprinted with permission from ref. 37. Copyright 2016 American Chemical Society. Further permissions related to the material excerpted should be directed to the ACS. Jayakannan and coworkers studied

38

oligo-phenylenevinylene (OPV)-containing amphiphilic

polyesters which formed highly luminescent nanoparticles owing to the strong aromatic π-π stacking. Stacked OPV dyes, present in the core of the particles, served as FRET donor for the noncovalently sequestered Nile red dye and these FRET particles were found to be highly promising for intra-cellular delivery and imaging in cancer cells.

Figure 16: Schematic showing FRET from stacked OPV to Nile red guest and lack of it for the isolated OPV chains. Reprinted with permission from ref. 38. Chemical Society.




FRET to study supramolecular polymers and assemblies: Supramolecular assembly enables creating wide-ranging nano-structures by weak and reversible non-covalent interactions.39-41 In the recent past, the focus has been shifted to supramolecular assemblies in aqueous medium with the objective of mimicking the structure and function of biological systems and making new biomaterials.42 Plenty of examples exist in Nature on complex non-covalent assemblies which are highly dynamic and that in turn controls their elegant functions. Albeit much progress in design and function of wide ranging supramolecular systems, relatively less information is known on the dynamics of these structures, although there is a common perception that these non-covalent structures are dynamic in nature. This part of the review describes the utility of a FRET in probing the dynamics of supramolecular polymers and related information on supramolecular assemblies. Meijer and coworkers studied 43 equilibrium dynamics of supramolecular polymers derived from 1, 3, 5-benzenetricarboxamides (BTAs)-derived building blocks (Figure 17) in water using FRET. A well-studied BTA-monomer was co-assembled with small amount of FRET donor or acceptor appended BTA derivative and these two separate samples were physically mixed. By monitoring the time dependent evolution of FRET, the equilibrium dynamics could be estimated in similar fashion that has been described for covalent polymers in the previous section. Time required for mixing (as evident by the saturation of the FRET ratio) was in hours indicating a comparable dynamics with that of the covalent polymers aggregates. In this interesting study they showed that the introduction of the homochirality resulted in the formation of a much robust structure as in this case it took significantly longer time (>20 h) for the FRET to saturate compared to that observed (2h) in the supramolecular polymer of the achiral system. Noteworthy that for both chiral and achiral systems, SAXS and TEM studied revealed identical self-assembly and structural features but by FRET studies the dynamics was found to be vastly different. Furthermore, the kinetics of the


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exchange in both systems could be analyzed by a biexponential decay revealing that a slow and a fast process were involved in the overall dynamics. The origin of such behavior though was not fully elucidated in this study.

Figure 17: a) Schematic showing equilibrium exchange of the FRET D and A dyes between separately labeled supramolecular polymers of BTA; Time dependent FRET ratio (originates from co-localization of the D and A dyes in the same polymer chain) of the b) achiral and c) chiral BTA supramolecular polymers showing significantly slow dynamics in the later case. Reprinted with permission from ref. 43. Copyright 2015 Springer Nature.

However, it is noteworthy that such observation was also made for guest exchange in amphiphilic polymer aggregates and thinking in similar line it is possible that (i) diffusion of the D/A dyeappended BTAs and (ii) collision of the polymers facilitating the exchange could be the possible two distinct processes involved in the overall exchange in this supramolecular polymeric system. Subsequently the same group showed44 the effect of the hydrophobicity on the supramolecular 25 ACS Paragon Plus Environment


polymerization and exchange dynamics in similar BTA-derives building blocks using FRET between the same D-A pair. It was concluded that a minimum of C11 alkyl chain was required to have an adequate hydrophobic shielding of the central BTA core to polymerize in water by Hbonding. For C11 and C12 chains, the exchange dynamics was comparable while BTA-derivative with C10 chains produced irregular results indicating lack of regular polymerization. In similar system, they further showed the utility of FRET to probe the control on the monomer sequence by a biological agent.45-46 Basically cationic BTA derivatives attached with either FRET donor or acceptor chromophore were co-assembled with the non-ionic non-fluorescently labeled BTAs (Figure 18).

Figure 18: Chain walking of D/A labeled cationic BTA derivatives in the supramolecular copolymer with non-ionic BTA derivatives in presence of anionic DNA of appropriate length resulting in close proximity of the D and A dyes and FRET which disappears in presence of an enzyme that destroys the multivalent interaction with the DNA. Reprinted with permission from ref. 45. Copyright 2015 The Royal Society of Chemistry.

These copolymers showed a negligible FRET as the average distance between D/A labeled building blocks was large in the statistically distributed copolymer. However in presence of an anionic single 26 ACS Paragon Plus Environment

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stranded DNA (ssDNA), the cationic BTA units showed clustering to avail the multivalent binding. It resulted in a much enhanced FRET as the cationic units were the ones which were labeled by the D/A fluorophore. In fact such clustering effect, as elucidated by the FRET studies, revealed a strong dependence on the length of the ssDNA which acted as a recruiter. Interestingly, in presence of an enzyme that degraded the DNA, the FRET ratio decreased and reached its previous value as the monomers again re-distributed along the entire polymer chain due to its dynamic exchange. They further investigated the kinetics of exchange of supramolecular polymers, assembled by selfcomplementary H-bonding between ureidopyrimidinone (UPy) groups in water.47

Figure 19: Supramolecular assembly of UPy attached polymers and their structure dependent dynamics probed by FRET studies. Reprinted with permission from ref. 47. Copyright 2017 The Royal Society of Chemistry.

Two types of polymers (attached with a single or two UPy groups in the hydrophobic terminal(s) of the polymer) (Figure 19) and their mixtures were investigated in context of the structure and dynamics. Donor Cy3 and acceptor Cy5 (FRET pair) were attached with these building blocks separately so that the exchange dynamics could be studied by FRET. These building blocks produced supramolecular polymers in water by quadruple H-bonding between the UPy groups and then laterally stacked by the H-bonding among the urea groups. Supramolecular polymers constructed from the building blocks containing two UPy units showed very fast exchange while those containing single UPy-attached building blocks showed remarkably slow dynamics. 27 ACS Paragon Plus Environment


Furthermore in the co-assembly of these two types of building blocks, the exchange dynamics could be tuned to a great extent. These results indicate a great opportunity to tune the dynamics of supramolecular polymers by judicial structure engineering. We have recently reported molecular interaction driven assembly of supramolecularly engineered amphiphilic polymers48-49 (Figure 20) in which the nature of the single H-bonding unit (hydrazide or amide), present in the terminal supramolecular structure directing unit, decides the morphology (polymersome or spherical/ cylindrical micelle, respectively) of the aqueous assemblies of the polymers.

Figure 20: a) Structure of supramolecular engineered amphiphilic polymers labeled with FRET D and A chromophores and b) their self-sorting probed by FRET in water. Reprinted with permission from ref. 48. Copyright 2017 John Wiley and Sons.

We were curious to examine the assembly pattern in their mixture. To this end, P1-50 was labeled separately with a red-emitting and a green-emitting dye resulting in P1-50-R and P1-50-G, respectively. The red and green dyes were chosen in such a way that they form a FRET pair. On the other hand P2-50 was labeled with only the red-emitting dye resulting P2-50-R. Pre-formed


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aggregates of P1-50-R and P1-50-G, when mixed in equal volume, resulted in an intense FRET suggesting their mixed assembly indeed brought the donor and acceptor chromophores within adequately close proximity for FRET. However, when the hydrazide containing P1-50-G and amide containing P2-50-R were mixed, negligible FRET was noticed which did not change over time indicating these two different aggregates did not mix together. This is remarkable considering that these two polymers differ only in the single H-bonding group present in the terminal of the chain and still they could form self-sorted assembly in aqueous solution. Sijbesma and coworkers probed the chirality driven self-sorted assembly of chiral trans-1,2bisureido cyclohexane-based bolaamphiphiles.50 (Figure 21).

Figure 21: Structure of chiral bola-amphiphiles together with FRET D/ A dyes which were used for labeling the supramolecular assembly. Due to the difference of the chirality of the linker USU and URU assembled in self-sorted manner which was probed by FRET between naphthalene and pyrene chromophores. Reprinted with permission from ref. 50.


Chemical Society.

Bola-amphiphiles USU and URU (Figure 21) differed only in the stereochemistry of the chiral cyclic linker which led to an enantio-selective supramolecular polymerization. They used pyrene /



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naphthalene labeled derivatives of these building blocks which were co-assembled and then selfsorting was tested by FRET. Later the same chiral linker was exploited by George and co-workers for enantio-selective supramolecular polymerization of core-substituted NDI building blocks (Figure 22) which was probed by the FRET technique.51 NDI chromophores are known to exhibit different photophysical properties depending on the nature of the core substitution.

Figure 22: a) Structure of cNDI derivatives with chiral linker and b) their chirality recognition in supramolecular polymerization which could be probed by FRET. Reprinted with permission from ref. 51. Copyright 2017. John Wiley and Sons.

They studied oxygen and amine substituted NDI derivatives as monomeric units which form the FRET D-A pair. These monomers differed only in the stereochemistry of the chiral spacer. In case of matching stereochemistry of the spacer, a mixed stack was formed resulting in FRET while in case of mismatch in the stereochemistry of the spacer, self-sorted stacks were formed and consequently the FRET was negligible. Around the same time, another report

52

showed enantio-

selective light harvesting in the co-assembly of similar diethoxy-substituted NDI molecule with 30 ACS Paragon Plus Environment

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perylenediimide. From the energy transfer data, it was concluded that the R- analogues preferentially recognized the oppositely configured (S) guests with stereo-selectivity of nearly 100%. While most of the systems described so far elucidate H-bonding driven supramolecular assembly, recently, Yang and co-workers53 reported an example where FRET could be highly useful in real time monitoring of the dynamics of co-ordination driven self-assembly of complementary building blocks attached with FRET donor (Coumarin) and acceptor (Rhodamine) molecules (Figure 23). Important features of such co-ordination driven self-assembly such as ligand exchange, anion induced disassembly, stability, solvent effect and reassembly could be precisely probed by monitoring the FRET between the D and A dyes which could come to a close proximity required for FRET only in the assembled state.

Figure 23: Schematic showing coordination driven cyclic polymerization of D/A appended ligands and emergence of FRET. Reprinted with permission from ref. 53. Copyright 2017 American Chemical Society.

In a recent report,

54

Schenning and co-workers have studied co-assembly behavior by FRET in

two sets of fluorene co-oligomers containing either naphthalene (D) or benzothiadiazole (A) dyes in the backbone (Figure 24). The effect of the polarity of the side chains on the stability, dynamics and self-sorting could be elucidated by FRET studies (Figure 24). They observed that 31 ACS Paragon Plus Environment


with apolar side chains, no exchange occurred between the self-assembled structures. Whereas, with polar side chains the exchange occurred at room temperature and with mixed chains, selfsorted domains in the same nanostructures or self-sorted separate nanoparticles were formed.

Figure 24:Top-Structure of fluorene co-oligomers with varying peripheral wedge; Bottom-Structure dependent co-assembly behavior as revealed by FRET between the co-monomer attached in the backbone. Reprinted with permission from ref. 54. Copyright 2013 American Chemical Society.


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SUMMARY AND OUTLOOK In this short review, we have highlighted the utility of the FRET as an excellent analytical tool to probe the dynamic exchange phenomenon in amphiphilic polymeric aggregates and supramolecular polymers in aqueous medium. We also have collated related examples those demonstrate that various other important information such as critical aggregation concentration, aggregation number, non-covalent encapsulation stability, structural integrity in biological and intra-cellular environment and the nature of the co-assembly features in supramolecular polymerization of multiple building blocks could be unambiguously probed by FRET studies. Although, FRET depends on a set of few rigid parameters, like distance dependence, orientation between the D and A, and the overlap of the donor emission and the acceptor absorption, it offers several advantages like sensitivity, applicability in dilute solution, precision in calculating distance between two points, apart from the easy commercial availability of the fluorescent dyes. By virtue of these, FRET becomes an unparallel technique to probe exchange dynamics, mechanism of exchange of amphiphilic polymer aggregates even in intra-cellular environment and also helps to track the location of a FRET aggregate (micelle co-encapsulated with FRET DA pair) in cellular compartments. However among large number of reported amphiphilic polymeric aggregates,11-12 only a few selective examples elucidate the exchange dynamics or non-covalent encapsulation stability. Amphiphilic branched polymers, dendrimer, star polymer and other architectures have hardly been studied in context of their exchange dynamics. Effect of structural parameters such as block length, hydrophobic/ hydrophilic balance, backbone functionality or effect of morphology (for example spherical vs. cylindrical micelle) on the exchange dynamics has also not been studied systematically. As discussed in this review,33-34 structural difference in the polymer can lead to a fully contrasting situation with regard to the



dynamic exchange behavior of the aggregate, particularly under biological conditions. Therefore, it is imperative to develop a thorough understanding on the impact of structural and other parameters on the chain exchange dynamics for designing future biomaterial and in this context FRET appears to be an excellent and easily employable experimental tool, for both in vitro and in vivo studies. While FRET has been employed to study amphiphilic polymeric aggregates for past 30 years, it is only recently that it has been used to study the dynamics of supramolecular polymers in similar way. These recent studies demonstrate plenty of opportunities to fine tune the structural parameters in the building block to modulate the exchange dynamics to a great extent. However only a handful of examples have been reported so far regarding exchange dynamics in supramolecular systems. Considering the increasing interest in synthetic supramolecular systems in water medium and their relevance as promising biomaterial, FRET studies may emerge in a big way to elucidate exchange dynamics in supramolecular systems.

AUTHOR INFORMATION Corresponding Authors *E-mail: Suhrit Ghosh: [email protected]; Priya Rajdev: [email protected] NOTES The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS SG thanks the DST, India for funding through the SwarnaJayanti Fellowship (DST/SJF/CSA01/2-14-15). PR thanks the Technical Research Centre for Molecules and Materials (DST), IACS, for a research fellowship and DST, India (SR/WOS-A/CS-97/2011) for funding.


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AUTHOR BIOGRAPHIES

Professor Suhrit Ghosh was born in 1976 in India. He completed MS (Chemistry) in 2000 from the Indian Institute of Science, Bangalore and continued there for PhD till 2005. He then moved to the University of Massachusetts, Amherst, USA as a post-doctoral research associate during 20052007 and subsequently worked in the University of Würzburg, Germany as an Alexander von Humboldt Postdoctoral Fellow. He returned to India in 2008 and joined the Indian Association for the Cultivation of Science Kolkata as an Assistant Professor where he currently holds the position of a Professor. Research interest of his group is focused on the supramolecular assembly of π-systems and macromolecules by directional interaction and stimuli responsive amphiphilic polymers.

Dr. Priya Rajdev obtained M. Sc. (Physical Chemistry) in 2003 from the University of Burdwan, India and completed PhD from the Indian Institute of Science, Bangalore in 2008. At present, she is a senior Research Associate at the Indian Association for the Cultivation of Science, Kolkata. Her research interest is in the Physical and Biophysical Chemistry of polymeric and supramolecular systems.


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