Insights into Noncovalently Core Cross-Linked Block Copolymer Micelles by Fluorescence Resonance Energy Transfer (FRET) Studies Priya Rajdev,* ,† Dipankar Basak, † and Suhrit Ghosh Polymer Science Unit, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Kolkata, India 700032 * S Supporting Information ABSTRACT: Amphiphilic polymers have emerged as an important class of materials owing to their ability to produce a diverse range of self-assembled structures with container properties that can be used to address growing challenges in biomedical applications. Thus, in-depth understanding on their aggregation properties is important fundamentally as well as from an application viewpoint. In this article we describe utilization of fluorescence resonance energy transfer (FRET) as a powerful tool to elucidate various physical properties of amphiphilic block copolymer aggregates at very low polymer concentration (∼10 -7 M) which otherwise are difficult to achieve using other commonly used techniques such as microscopy, scattering, or external probe based spectroscopic techniques. We synthesized a prepolymer based on PEO-b-PMMA-co-PHEMA and subsequently utilized the hydroxy groups of the HEMA units to covalently attach with either a green (D) or a red (A) fluorescent dye (D-A pair suitable for FRET) to get D- and A-labeled amphiphilic polymers with similar spatial distribution of these chromophores in the hydrophobic block of the two polymers. Coassembly of red and green labeled polymers in micellar aggregates results in highly efficient FRET while no FRET is observed when they remain as unimer. This was exploited to study the micellization process by solvent, concentration, time, and pH dependent FRET studies either by mixing preformed aggregates of the two polymers or by inducing aggregation in mixture of unimers. Contrary to existing perception, our studies revealed exceptionally slow dynamics (mixing time >50 h), very low critical aggregation concentration (<10 -7 M), remarkably high tolerance to good solvent, and intriguing solvent induced swelling followed by disassembly of the micellar aggregate from this rather simple diblock copolymer. We explicitly show why the FRET based tool stands out among other techniques to probe such detail physical characteristics of micellar aggregates with precision at very dilute concentration which otherwise appears to be a rather difficult task. More interestingly, we provide the rationale behind the exceptionally high stability of these micelles by providing evidence of noncovalent core cross-linking by H-bonding among the few unreacted OH groups present in the hydrophobic block. When this parameter was lifted off by either protonation at acidic pH or protecting the hydroxyl group by acetyl group, FRET studies showed very fast dynamics of the micellar aggregations, confirming the free OH groups in the hydrophobic domain are responsible for noncovalent core cross-linking leading to the unusual stability of the micelles. ■ INTRODUCTION Aggregation of amphiphilic polymers (particularly block copolymers) to various structures such as micelle, polymer- some, and others 1-5 have been studied with great interest owing to their close relevance in biomedical application, 6-11 including drug delivery, 12-16 gene delivery, and sensing. 17-20 Hence, precise estimation of physical parameters such as CAC (critical aggregation concentration), morphology, dynamics, particle size, or stability of these aggregates is of utmost importance for their biological applications. Most of the common techniques used to probe polymer aggregates 21 in solution involve either scattering or microscopy which primarily provides information on the static properties of the assemblies but not the dynamic behavior. However, estimation of the dynamic nature of a given aggregate is very important, as it is related to undesired leakage of the encapsulated guest/drug molecules. In this context spectroscopic techniques are more reliable. Most commonly used spectroscopic technique to probe amphiphilic polymer aggregates is fluorescence by noncovalently encapsulating fluorescent probe molecules in the aggregates and studying their spectroscopic signature which is often different when they are located in the hydrophobic confined domain. This approach, although having been used in many systems and providing adequate information, still lacks the precision in estimating important physical parameters as it is associated with issues like partitioning of the dye molecules between the aggregates and solution. This is particularly true at dilute polymer solution and also in the context of studying dynamics of polymer aggregates by fluorescence resonance energy transfer (FRET) 22-31 for which instead of a single dye a pair of suitable donor (D) and acceptor (A) fluorophores need to be coencapsulated. The other approach is covalently linking Received: March 16, 2015 Revised: April 24, 2015 Article pubs.acs.org/Macromolecules © XXXX American Chemical Society A DOI: 10.1021/acs.macromol.5b00559 Macromolecules XXXX, XXX, XXX-XXX