Location of a Hydrophobic Load in Poly(oligo(ethylene glycol) methyl ether methacrylate)s (PEGMAs) Dissolved in Water and Probed by Fluorescence.

Two series of pyrene-labeled poly(oligo(ethylene glycol) methyl ether methacrylate)s referred to as PyEG5-PEGnMA and PyC4-PEGnMA were prepared to probe the region surrounding the polymethacrylate backbone by using the fluorescence of the dye pyrene. PyEG5-PEGnMA and PyC4-PEGnMA were prepared by copolymerizing the EGnMA methacrylate monomers with penta(ethylene glycol) 1-pyrenemethyl ether methacrylate or 1-pyrenebutyl methacrylate, respectively. In organic solvents, the much longer 18 non-hydrogen atom linker connecting the pyrene moieties to the polymethacrylate backbone in the PyEG5-PEGnMA samples enabled the deployment of the pyrenyl labels into the solution. In water, however, an excited pyrene for PyEG5-PEGnMA was found to probe a same volume as for the PyC4-PEGnMA samples where a much shorter 6 non-hydrogen atom spacer connected pyrene to the backbone. Another surprising observation, considering that the hydrophobicity of pyrene induces strong pyrene aggregation for many pyrene-labeled water-soluble polymers (Py-WSPs) in water, was the little pyrene aggregation found for the PyEG5-PEGnMA and PyC4-PEGnMA samples in water. These effects could be related to the organic-like domain (OLD) generated by the oligo(ethylene glycol) side chains densely arranged around the polymethacrylate backbone of the polymeric bottlebrush (PBB). Additional fluorescence experiments conducted with the penta(ethylene glycol) 1-pyrenemethyl ether derivative indicated that the cylindrical OLD surrounding the polymethacrylate backbone had a chemical composition similar to that of ethylene glycol. Binding of hydrophobic pyrene molecules to unlabeled PEGnMA bottlebrushes in water further supported the existence of the OLD. The demonstration, that PEGnMA samples form an OLD in water, which can host and protect hydrophobic cargoes like pyrene, should lead to the development of improved PEGnMA-based drug delivery systems.

Probing the inner local density of complex macromolecules by pyrene excimer formation.

The direct relationship existing between the average rate constant 〈k〉 for pyrene excimer formation and the local concentration [Py]loc of ground-state pyrenyl labels covalently attached to a macromolecule was established for 55 pyrene-labeled macromolecules (PyLM). These PyLM belonged to three different families of macromolecules with the first representing short monodisperse linear chains end-labeled with pyrene (polystyrene, poly(ethylene oxide), and poly(N-isopropyl acrylamide)), the second representing long polydisperse linear chains randomly labeled with pyrene (poly(methyl acrylate), poly(methyl methacrylate), polystyrene, poly(butyl methacrylate), poly(methoxyethyl methacrylate), and poly(N-isopropyl acrylamide)), and the third being comprised of two series of pyrene end-labeled low generation dendrimers with a bis(hydroxymethyl)propionic acid or a polyamidoamine backbone. The assumption, that the polymeric segments probed by an excited pyrenyl label covalently attached to one of these macromolecules obeyed Gaussian statistics, enabled the calculation of their square root average squared end-to-end distance (LPy), which was applied to calculate [Py]loc. The log-log plots of 〈k〉 as a function of [Py]loc yielded straight lines with a slope of unity for all families of macromolecules studied in four different organic solvents demonstrating the validity and generality of the 〈k〉-vs.-[Py]loc relationship. Since an experimentalist knows how the the pyrenyl labels are covalently attached onto a macromolecule, [Py]loc offers a means to probe the local density of a macromolecule, which can be employed to characterize its conformation in solution. Consequently, the 〈k〉-vs.-[Py]loc relationship provides a novel experimental means to probe the conformation of macromolecules which should establish pyrene excimer formation as an appealing method for conformational studies of macromolecules in solution, which should nicely complement scattering techniques.

Simplification in the Acquisition and Analysis of Fluorescence Decays Acquired with Polarized Emission for Time-Resolved Fluorescence Anisotropy Measurements.

This study introduces a global fluorescence decay analysis that substantially simplifies the acquisition and analysis of time-resolved fluorescence decays acquired with a vertically polarized excitation and vertically (IVV(t)) and horizontally (IVH(t)) polarized emission for time-resolved fluorescence anisotropy (TRFA) measurements. TRFA measurements were conducted whereby the IVV(t) and IVH(t) fluorescence decays of a series of oligoquinolines labeled at one end with an oligo(phenylenevinylene) dye (OPV-Qn with n = 4, 7, 17, 24, 33) were acquired according to the standard protocol that is currently accepted in the scientific literature which involves toggling the emission polarizer before fitting linear combinations of the IVV(t) and IVH(t) decays or acquiring the IVV(t) and IVH(t) decays with static polarizers before fitting them globally. The rotational time (ϕ) and initial anisotropy (r0) retrieved from these analyses were identical within experimental error regardless of whether the decays were acquired with toggling or static polarizers and fitted according to the standard protocol or globally. These experimental results were further supported by retrieving the parameters used to generate mono-, bi-, and tri-exponential TRFAs from the global analysis of simulated IVV(t) and IVH(t) fluorescence decays which were found to match perfectly the values that were inputted. Together, these experiments and simulations demonstrated that the parameters describing any type of TRFA can be extracted directly from the analysis of the IVV(t) and IVH(t) fluorescence decays acquired with a standard time-resolved fluorometer, a substantial simplification compared to the protocols currently in place to determine the TRFA.

Accurate Determination of the Average Rate Constant of Pyrene Excimer Formation for Pyrene-Labeled Macromolecules from the Analysis of Individual Fluorescence Decays with Sums of Exponentials.

The average rate constant (⟨k⟩) for pyrene excimer formation (PEF) between an excited and a ground-state pyrenyl label covalently attached to a pyrene-labeled macromolecule (PyLM) has been found to be an ideal parameter to probe macromolecular conformations due to its proportionality to the local concentration ([Py]loc) of pyrenyl labels in PyLM. To date, ⟨k⟩ has only been determined with the model-free analysis (MFA) involving the global analysis of the pyrene monomer and excimer fluorescence decays of PyLM. Unfortunately, the MFA is computationally demanding which prevents its widespread use. To circumvent this complication, a methodology is introduced that involves the analysis of individual fluorescence decays with sums of exponentials (SoE), which are commonly used in the analysis packages of commercial time-resolved fluorometers. The individual fluorescence decays of the pyrene monomer acquired with 286 PyLM were analyzed with a SoE to yield ⟨kSoE-M⟩. The strong correlation between ⟨kSoE-M⟩ and ⟨kMF⟩ obtained from the global MFA indicated that ⟨kSoE-M⟩ was a good representation of ⟨k⟩. Furthermore, the AE-/AE+ ratio, equal to the ratio of the sum of the negative pre-exponential factors over the sum of the positive pre-exponential factors, was determined by fitting the individual pyrene excimer fluorescence decays of the 286 PyLM with a SoE. AE-/AE+ was found to take a value between -1.0 and -0.8, indicating that the pyrenyl labels were not aggregated. This result indicated that [Py]loc was well described by ⟨kSoE-M⟩, so that ⟨kSoE-M⟩ could be used to describe the conformation of macromolecules in the same manner as ⟨kMF⟩. Consequently, the methodology based on the analysis of individual fluorescence decays with sums of exponentials to determine ⟨kSoE-M⟩ and AE-/AE+ provides a robust alternative to the use of the MFA for the study of PyLM to many scientists interested in the characterization of macromolecular conformations.

Flexibility of Poly(alkyl methacrylate)s Characterized by Their Persistence Length Determined through Pyrene Excimer Formation.

A series of poly(alkyl methacrylate)s and poly(oligo(ethylene glycol) methyl ether methacrylate)s labeled with 1-pyrenebutanol were referred to as the PyC4-PCnMA samples with n = 1, 4, 6, 8, 12, and 18 and the PyC4-PEGnMA samples with n = 0-5, 9, 16, and 19, respectively. Pyrene excimer formation (PEF) upon the encounter between an excited and a ground-state pyrenyl labels was employed to determine their persistence length (lp) in o-xylene. The fluorescence decays of the PyC4-PCnMA and PyC4-PEGnMA samples were acquired and analyzed with the fluorescence blob model to yield the number (Nblob) of structural units in the volume probed by an excited pyrenyl label. Nblob was found to decrease with an increasing number (NS) of non-hydrogen atoms in the side chain, reaching a plateau for the PyC4-PEGnMA samples with a longer side chain (n = 16 and 19). The Nblob values were used to determine lp. The lp values for the PyC4-PCnMA and PyC4-PEGnMA samples increased linearly with increasing NS2 as predicted theoretically, which agreed with the lp values obtained by viscometry for a series of PCnMA samples. The good agreement between the lp values retrieved by PEF and viscometry served to validate the PEF-based methodology for determining lp for linear polymers.

Persistence Length of PEGMA Bottle Brushes Determined by Pyrene Excimer Fluorescence.

Seven pyrene-labeled poly(oligo(ethylene glycol) methyl ether methacrylate)s (PyEG5-PEGnMAs) were prepared with n = 0, 3, 4, 5, 7, 9, and 19 ethylene glycol units by copolymerizing a small amount of penta(ethylene glycol) 1-pyrenemethyl ether methacrylate with an EGnMA monomer. The conformation of the PyEG5-PEGnMA polymers evolved from a random coil for PyEG5-PEG0MA or poly(methyl methacrylate) to a polymeric bottle brush (PBB) architecture with increasing side chain length. The fluorescence decays of the PyEG5-PEGnMA samples were fitted according to the fluorescence blob model (FBM) whose parameters were used, in combination with the Kratky-Porod equation, to calculate the persistence length of these polymers. The persistence lengths obtained from the PEF experiments were found to increase with the square of the number (NS) of non-hydrogen atoms in the side chain as expected theoretically. The persistence lengths found with the PyEG5-PEGnMA samples in DMF also matched those found earlier for another series of PEGnMA samples labeled with 1-pyrenebutanol. The good agreement found between the persistence lengths obtained with the PEGnMA samples labeled with two different pyrene derivatives illustrates the robustness of the method and its applicability for measuring the unknown persistence length of polydisperse polymer samples.

Characterization of the Distribution of Pyrene Molecules in Confined Geometries with the Model Free Analysis.

Evidence is provided showing that global Model Free Analysis (MFA) of monomer and excimer fluorescence decays of pyrene dissolved in aqueous solutions of sodium dodecyl sulfate (SDS) provides the same structural and dynamic information on SDS micelles as the well-established Micelle Model (MM) does. Both MFA and MM were employed to characterize the quenching kinetics between dyes and quenchers located in surfactant micelles and the aggregation number of surfactant micelles. However, contrary to the MM, which assumes that dyes and quenchers distribute themselves among SDS micelles according to a Poisson distribution and react with a rate constant that is proportional to the number of reactants in a micelle, the MFA accomplishes this task without making any assumption about the process of pyrene excimer formation in SDS micelles. The ability of the MFA to retrieve accurately the molar fraction of pyrene molecules that are isolated in SDS micelles and do not form excimers was taken advantage of to establish that it equaled the Poisson probability of exciting micelles that contained a single pyrene. The molar fraction of isolated pyrenes could then be utilized to determine the aggregation number of the SDS micelles, and the rate constant of excimer formation between one excited- and one ground-state pyrene located inside a same micelle. Within experimental error, both the MFA and MM yielded the same micelle aggregation number and rate constant of excimer formation, with the MFA making no prior assumptions about the physical principles underlying the process of excimer formation contrary to the MM. The ability of the MFA to retrieve quantitative parameters providing structural and dynamic information about macromolecular systems with no prior knowledge about their architecture or labeling scheme implies that it can be applied to characterize a wide range of macromolecular architectures in solution.