Hyaluronidase can be presented to the target tissue in a multivalent display by using nanotechnology
to synthesize nanosized water dispersible nanostructures which are later derivatized with the enzyme
. The use of nanotechnology provides a controllable and versatile approach to construct different systems at will, of different sizes and shapes and provides control over the way the enzyme is presented on the surface. Through "wet", bottom-up synthesis, one can produce different shaped nanostructures, like elongated ones, which are known to enhance the affinity for the target compared to the spherical shape [46
]. Hyaluronidase may be attached to the surface of the nanostructure by chemical coupling or using the layer-by-layer self-assembly technique.
Multivalent interactions of Hyal with the target tissue and a lower diffusion coefficient of the nanosized object are expected to provide a longer residence time of Hyal in contact with the substrate and result in a more effective degradation of HA.
To demonstrate that the multivalent presentation of Hyal is indeed more effective than using the free enzyme, Scodeller et al. have described the first nanoformulation of Hyaluronidase [17
]. In that work, melanoma
tumors were pretreated with solutions of equal enzymatic activities of free and immobilized Hyal, and the authors showed that the immobilized Hyaluronidase was more effective than the free enzyme for their treatment study. The degrading properties of these particles showed to be quite dramatic when analyzing the tissues under Field Emission-Scanning Electron Microscopy (FE-SEM) even after only one application of the Hyal-modified nanoparticles (Figure 1A and B)
In that work, human melanoma
A375 inoculated tumors were treated with Carboplatin, with the free and immobilized Hyal pretreaments using peritumoral injections. To functionalize these particles with Hyal the authors used the layer-by-layer (LBL) self-assembly technique [48
] which consists of the alternate adsorption of polycations and polyanions from water solutions, where each layer deposition is accompanied by charge reversal as a result of charge overcompensation [49
]. The LBL technique was first used by Caruso et al. to successfully functionalize micrometer sized colloidal surfaces with enzymes, preserving enzymatic activity [50
], and the group of Calvo was the first to successfully functionalize nanometer colloidal gold surfaces with enzymes, conserving catalytic activity [51
This LBL technique is the most appealing when functionalizing surfaces with enzymes since enzymatic activity is maintained and multiple layers may be deposited to yield a larger response; this method can be employed to construct catalytically active enzymatic multilayers by self-assembling enzymes and polymers on planar [52
] and colloidal surfaces [51
]. The catalytic activity of this multilayer will depend on enzymatic loading, enzyme orientation and mobility, parameters which are directly controlled by the deposition conditions (assembly pH and ionic strength) [53
]. Also, importantly, many times the specific enzymatic activity (enzymatic activity/moles of enzyme) is increased upon surface immobilization. Monteiro et al. [54
] have immobilized Hyal on Langmuir Blodgett films prepared with the sodium salt of dihexadecylphosphoric acid, ending with dipalmitoylphosphatidylcholine. In that study, tests of enzymatic activity, using Hyaluronic acid as the substrate, showed an increase of activity compared to the homogeneous medium and the authors propose a model of protein insertion with Hyal-lipid chains hydrophobic interactions and the hydrophilic site containing the active site, exposed to the solution.
The particles used by Scodeller et al. are 250 nm sized silica spheres, of low cost and ease to synthesize and which have proved to be biocompatible [55
]. Currently, a fluorescent version of this same system is being studied, in which silica nanoparticles are replaced with a fluorescent Fluorescein core protected by a shell of silica matrix [56
] which provides a very photostable signal and allows for long term live imaging of these particles [57
In this nanoformulated Hyal, a smaller diffusion coefficient and multivalent interactions (affinity scales exponentially with number of valences) with the target tissue is the proposed mechanism to explain the enhanced adjuvant effect respect to free Hyal.