Controlling aggregation of Rose Bengal with nanocages to modulate the production of Reactive Oxygen Species in the context of Photochemical Tissue Bonding

Abstract

The healing of wounds has always been a complicated matter because suturing, the long-standing closure method, depends on the skill of the surgeon, relies on placing a foreign body in the tissue which makes it prone to inflammation and infection, and leads to scarring and, most often, lower mechanical properties of the tissue. For instance, mortality directly attributable to surgical site infections goes up to 2.5% per year in USA. Some alternative methods for the closure and treatment of wounds and surgical incisions have been studied, where one of the newest alternatives is Photochemical Tissue Bonding (PTB). PTB involves applying a photosensitive molecule, Rose Bengal (RB), to the wounded site and then exposing it to visible light (~𝟓𝟑𝟎 𝒏𝒎). Despite its proven benefits, the reaction mechanism involved in enhanced crosslinking of collagenous tissue remains unclear. It is important that we know how RB behaves to adjust the protocol of the therapy and increase its efficiency. It is believed that this process is mediated by singlet oxygen, which leads to the production of Reactive Oxygen Species (ROS). Furthermore, it is suspected that the aggregation of RB negatively affects the photon-efficiency of RB. However, a comprehensive reaction mechanism accounting for RB aggregation and photochemistry in tissue remains elusive. In this thesis, we analyzed the aggregation of RB in phosphate buffer saline (PBS) aqueous solutions and studied the effect of its dimerization on the photobleaching and oxygen consumption kinetics. To study the thermodynamics of aggregation we utilized UV-Vis absorbance spectroscopy and DLS. It was confirmed that the dimerization constant for RB in 1X PBS is in the order of 1.1 x103 M. We confirmed that at a concentration of 1mM RB is found in a 1:1 monomer/dimer ratio. Then, we compare the extent of dimerization in the absence and presence of three nanocages: cucurbit[7]uril, β-cyclodextrin and hydroxypropyl-γ-cyclodextrin (HP-γ-CD). HP-γ-CD is the nanocage that is best suited to bind to RB. Host-guest complexation of RB with HP-γ-CD appears to delay dimerization of RB in 1X PBS significantly. We then correlated aggregation to photochemical kinetics. Steady-state kinetics were tested with real-time absorbance spectroscopy and fluorescence-based oxygen monitoring. Evidence was obtained indicating that the complexation of RB with HP-γ-CD can increase the rate of consumption of molecular oxygen under irradiation. We therefore conclude that is appears possible to use an appropriate nanocage to form host-guest complexes that delay dimerization of RB is aqueous PBS solutions, and that the caging of certain moieties in the solvated RB may enhance de consumption of molecular oxygen.