Aggregation behavior of protoporphyrin IX in aqueous solutions:: Clear evidence of vesicle formation

The aggregation behavior of protoporphyrin IX in aqueous solution, as a function of pH and ionic strength, has been studied by means of UV/vis, fluorescence emission spectroscopy, and resonant light scattering (RLS) techniques. Our experimental results agree with previous literature assignments: (i) protoporphyrin IX is a monomer in the pH range 0-3, (ii) a dimer is present for pH > 8, and (iii) higher aggregates are present in the pH range 3-7. Addition of sodium chloride up to 0.3 M to a porphyrin solution at pH 12 gives a process resembling a phase transition, whereas it has little effect on acidic or neutral solutions. The apparent split Soret band observed in the intermediate pH range has been explained using a model in which dimers of porphyrins (with a slip angle alpha = 38degrees or 52degrees, as derived from depolarized RLS measurements) are the basic units and they interact axially through pi-pi stacking and laterally by edge-to-edge hydrophobic contacts. The half neutralization of the carboxylic acid side chains is responsible for the occurrence of a network of intermolecular hydrogen bonds, which contribute to a better stabilization of the supramolecular assembly. Evaporation on a glass surface of solutions containing protoporphyrin IX aggregates from samples at intermediate pH leads to aggregates stable enough to be investigated for the first time through scanning electron (SEM) and scanning near-field optical microscopy (SNOM). These species evidence a prolate shape with an average size of 200-500 nm and a medium height of 60 nm, which is in agreement with the hydrodynamic radii as measured in solution by dynamic light scattering. On consideration of the amphiphilic character of protoporphyrin IX, these observations suggest the formation of multilamellar or onion-like vesicles. The analysis of the SNOM images points to the presence of regions in which the aggregation process resulted in a thin film covering the vesicles. An analysis of SEM experiments reveals also the contemporary presence of large regions in which the vesicles collapse in a continuum layered structure.