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Core Assisted Formation of Porphyrin J-Aggregates in pH Sensitive Polyelectrolyte Microcapsules Followed by Fluorescence Lifetime Imaging Microscopy Vanda Vaz Serra, Nuno G. B. Neto, Suzana M Andrade, and Silvia M.B. Costa Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01390 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017
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Core Assisted Formation of Porphyrin J-Aggregates in pH Sensitive Polyelectrolyte Microcapsules Followed by Fluorescence Lifetime Imaging Microscopy Vanda Vaz Serra, a,b* Nuno G. B. Neto,a Suzana M. Andrade,a Sílvia M. B. Costaa a) Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal. b) Unidade de Química Orgânica e Produtos Naturais, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal.
KEYWORDS: Polyelectrolyte Microcapsules; Core-shells; Porphyrins; Aggregates; Fluorescence Lifetime Imaging Microscopy (FLIM).
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ABSTRACT
A strategy assisted by an inorganic template was developed to promote the organized selfassembly of meso-(tetrakis)-(p-sulfonatophenyl)porphyrin (TPPS) on pH sensitive core-shell polyelectrolyte microcapsules (PECs) of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). A key feature of this strategy is the use of template CaCO3 microparticles as a nucleation site endorsing inside-outside directional growth of porphyrin aggregates. Using this approach, TPPS self-assembly in positively charged PECs with CaCO3 (PAH/PSS)2PAH as a sequence of layers was successfully achieved using pH mild conditions (pH 3). Evidence for porphyrin aggregation was obtained by UV-Vis with the characteristic absorption bands in PECs functionalized with porphyrins.
Fluorescence Lifetime Imaging Microscopy (FLIM) of
polyelectrolyte core-shell confirmed the presence of radially distributed needle like structures sticking out from polyelectrolyte shells. Microscopic images also revealed a sequential process (adsorption, redistribution and aggregation) for the directional growth (inside/outside) of TPPS aggregates and highlight the importance of the core in the aggregation induction. Removing the CaCO3 core alters the porphyrin interaction in PECs environment and aggregates growth is no longer favored.
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INTRODUCTION Polyelectrolyte capsules (PECs) are micro- or nanostructures of a polyelectrolyte shell surrounding an aqueous droplet (hollow PECs) of an organic or inorganic core (core-shell PECs). First introduced in the end of the last century, these systems were initially described as promising colloids for technological and biotechnological applications. Their intrinsic characteristics led to an explosive growth in scientific interest regarding its potential applications as drug delivery systems,
biosensors,
micro/nano
reactors,
artificial
cells
and
catalysis.1-4
Stability,
micro/nanometric size control, the possibility of using biocompatible constituents and controllable uploading/release of functional materials are commonly referred to as the most significant features associated to its widespread recognition. Hence, PECs are a promising matrix for the incorporation of functional materials such as porphyrinoids. Porphyrinoids are tetrapyrrolic compounds with four pyrrole rings connected by methine bridges. They are aromatic compounds with π electrons delocalized over the macrocycle plane and well defined absorption bands in the UV-Vis region: a Soret band near 410 nm and four Q bands in the absorption range 500-800 nm. These molecules, known as “molecules of life” since they participate in vital processes such as photosynthesis and oxygen transport and storage, have a natural tendency to self-assemble through weak intermolecular interactions such as π-πstacking, hydrogen bonds and van der Waals forces. Supramolecular assemblies of individual chromophores organized into highly hierarchical structures have been explored to construct biomimetic photosynthetic antenna systems foreseeing applications in energy transport for molecular-scale electronics, optical devices, light-energy conversion and catalysis. From those, porphyrin J-aggregates are considered to be an efficient
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model to mimic light harvesting systems, whereby chromophores act collectively supporting faster energy transport due to strong intermolecular coupling.5 Monomeric porphyrins can achieve different types of excitonic coupling: J-aggregates resulting from side-to-side coupling of transition dipoles, leading to a red-shift of the Soret and Q absorption bands of the porphyrin, and also H aggregates that are triggered by porphyrin face to face coupling (π-π stacking), and turning out in a blue shift in the Soret band. Currently, there is an increasing interest in the study of the role of H and J-excitonic bands in soft matter light harvesting systems of potential optoelectronic applications.6-8 In this context, the aggregation of organic dyes such as porphyrins and PECs can be promising partners. Concerning porphyrinoid ordered self-assembly into well-defined hierarchical structures, one of the most interesting is meso-(tetrakis)-(p-sulfonatophenyl)porphyrin (TPPS) which is an amphiphilic porphyrin that under proper pH and ionic strength can form H or J-aggregates. TPPS aggregates induced by templates including polyamidoamine dendrimers,9 polyamines,10 peptides,11 proteins,12-14 surfactants,15 ionic liquids,16 polyelectrolyte multilayer films17 and confined environments such as AOT reverse micelles18, vesicles19 and carbon based materials,20 are also described in literature. Guo et al
21
showed that the photocatalytic activity of porphyrin aggregates is dependent on
their morphology. A significant higher photocatalytic activity was observed in nanofibers of 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (ZnTPyP), than in nanospheres of the same porphyrin, both prepared with the same surfactant assisted self-assembly methodology. Bera et al22 found a 1.9 fold increment on the photocurrent generated under visible light illumination due to
electron
transfer
from
a
composite
system
made
of
5,10,15,20-tetrakis(4-
carboxyphenyl)porphyrin nanorods (TCPP NR) and reduced graphene oxide (RGO).
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Hayashi et al23 used a sol-methodology to prepare biocompatible and hydrophilic silicaporphyrin hybrid nanotubes, through π-π stacking and electrostatic interactions, from cationic organoalkoxysilane and an anionic porphyrin (TCPP). Its use as near infrared (NIR) probe for in vivo imaging was reported. As an extension of that work, the same team went a step forward and prepared NIR probes of ring-shaped silica nanoparticles with a high content of ring-shaped TCPP aggregates also for in vivo imaging.24 Previously, Sadavisan et al 25 studied the self-aggregation of a tetraanionic porphyrin into heat sensitive multilayers hollow microcapsules (HPECs) made of strong polyelectrolytes poly(diallyldimethylammonium chloride) and poly(styrene sulfonate) multilayers. In their approach, porphyrin self-assembly in polyelectrolyte microenvironment was promoted through sequential additions of salt and acid. Satellite like structures of HPECs containing cylindrical nanotubes protruding externally were isolated from the nanotubes formed in the bulk solution and further characterized by conventional spectroscopic and microscopic techniques (AFM, TEM and CLSM). The aggregate’s dimensions were found to be dependent on the temperature, but the surface charge of the microcapsules had no influence on the orientation of the nanotubes. Other few examples already reported in the literature of dyes aggregation in hollow polyelectrolyte microcapsules include 6-carboxyfluorescein and rhodamine 6G selective precipitation26 or pseudoisocyanine inclusion. 27 Considering the promising properties of self-assembled porphyrinoid systems and the advantages of micro- and nano-confinement, our interest was focused in the development of photoactive PECs through the functionalization of polyelectrolyte microcapsules with porphyrin aggregates.
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Herein, we describe in detail the optical properties of diacid TPPS (acid conditions) in coreshell PECs (CaCO3(PAH/PSS)2PAH) and with each one of core-shell PECs building blocks (negative PSS and positive PAH polyelectrolytes and the CaCO3 core microparticle), using absorption, steady state and time resolved spectroscopies. Results of associated fluorescence quenching, excited states average and individual molecule lifetimes which were followed by Fluorescence Lifetime Imaging Microscopy (FLIM) as a function of CaCO3 core, pH and surface composition were used to monitor TPPS porphyrin aggregation upon its adsorption on core-shell PECs. FLIM is a powerful technique that provides spatially resolved images of the fluorophore lifetime, i.e., reports on the fluorophore microenvironment independently from fluorescence intensity.28 In particular, FLIM shows evidence of growing porphyrin aggregates within coreshell PECs whereas in hollow PECs they are not formed highlighting the relevance of the core in this process. In this paper, we report a well succeeded formation of porphyrin J-aggregates within polyelectrolyte microcapsules PECs, by combining the physicochemical properties of an inorganic template (CaCO3), with those of a diacid porphyrin (H4TPPS-2) and of pH sensitive polyelectrolyte multilayers containing both strong and weak polyelectrolytes (PSS and PAH, respectively). This combination induces the in situ ordered directional growth of porphyrin J aggregates from the core to interface (inside out). A major advantage of such methodology is a single step procedure controlled exclusively within the polyelectrolyte structure.
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EXPERIMENTAL SECTION
Materials Poly (sodium 4-styrenesulfonate), PSS (MW~75000, 18% wt.% in water) and poly(allylamine hydrochloride), PAH (MW~15000) (Figure S1) were obtained from Sigma-Aldrich. TPPS was purchased from Fluka (ε 413 nm = 5.1 x 105 M-1 cm -1).(12) Trifluoroacetic acid (TFA) and sodium hydroxide (NaOH) were used in order to control pH and were purchased from SigmaAldrich. All reagents were used without additional purification. Polyelectrolyte solutions (3 mg/mL, 0.5 M NaCl) were prepared in bi-distilled water and pH adjusted at 6.5 (for PSS/PAH polyelectrolyte microcapsules preparation) or pH 1.5 and 3.0 (for titration measurements). Glass microscope slides (0.13 - 0.17 mm thickness) were obtained from Normax, Portugal.
Preparation of CaCO3 template CaCO3 microparticle templates were prepared according to procedures well described in the literature.29 Equal volumes of saturated solutions of Na2CO3 and CaCl2 (0.33 M) were mixed under intense stirring. After 1 minute, the mixture was left resting for 15 minutes. CaCO3 microparticles were obtained after supernatant removal followed by three washing/ centrifugation cycles.
Preparation of core-shell and hollow PSS/PAH polyelectrolyte microcapsules The pH responsive core-shell and polyelectrolyte microparticles were prepared as follows: CaCO3 microparticles were dispersed in an aqueous solution of PAH (3 mg/ml, 0.5 M NaCl). After stirring for 45 minutes, the resulting particles were centrifuged (6000 rpm, 10 minutes) and
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the supernatant was removed. The templates were washed with distilled water (10 ml) and collected. After three washing / centrifugation cycles to remove residual PAH, the obtained microparticles were dispersed in an aqueous solution of PSS (3 mg/mL, 0.5 M NaCl). Layer-bylayer adsorption of opposite charged polyelectrolytes was repeated until the deposition of the 5 polyelectrolyte layers. The preparation of hollow PECs was achieved by suspending core-shells with the desired number of polyelectrolyte layers in EDTA (10 mL, 0.1 M) to destroy the core and stirred for 30 minutes at room temperature. This process was repeated twice. The prepared microparticles were washed three times with distilled water before further use.
Porphyrin adsorption onto PECs microcapsules H4TPPS-2 adsorption was made onto the core-shell microparticles with PAH as the last layer CaCO3(PAH-PSS)2PAH. Typically, capsules suspension was added to a H4TPPS-2 solution (3 μM, pH 3.0, adjusted using TFA) and the system was kept resting at room temperature overnight. The supernatant was then carefully removed and the microcapsules washed three times with distilled water (pH 3.0). The doped core-shell PECs are stable during at least 1 month of preparation and storage in the dark.
Methods A PerkinElmer Lambda 35 spectrophotometer was used in UV-Vis absorption measurements. Corrected fluorescence measurements were recorded with a SPEX Fluorolog spectrophotometer (HORIBA Jobin Yvon). Excitation at 445 nm was achieved using a NanoLED (fwhm
