Onofrio Annunziata

Research Interests

My research focuses on understanding and controlling phase transitions and transport properties of macromolecular solutions relevant to biotechnology and medicine. These studies will involve techniques such as interferometry, static and dynamic light scattering, turbimetry, chromatography, dialysis and microscopy. Below I summarize some relevant aspects of my research objectives.

Proteins. Proteins in solution are subject to crystallization, liquid-liquid phase transition, self-assembly, aggregation and gelation. Understanding and controlling these processes is important for protein crystal growth, protein aggregation diseases and the processing and use of biomaterials. Fig. 1 shows the formation of protein crystals (A) and protein-enriched liquid droplets (B) generated from aqueous solutions. I am interested in understanding how different associative processes interact with each other. For example, protein aggregation and protein self-assembly compete with protein crystallization; this is a notorious problem when trying to produce protein crystals. Another interesting case is provided by the enhancement of protein crystal nucleation in the proximity of the phase boundaries of the liquid-liquid phase transition. All these associative mechanisms depend on several factors such as protein primary structure, protein concentration, temperature, protein net charge, and presence of additives (e.g. salts, polymers and surfactants). It is not well understood how these variables favor one transformation relative to another. One of my goals is to identify strategies that optimize protein crystallization with respect to other competing associative processes.

Figure 1. Crystallization (A) and liquid-liquid phase separation (B) of protein aqueous solutions.

Dendrimers. Dendrimers are a class of globular branched macromolecules (see Fig. 2), which may be used as uni-molecular micelles, drug delivery systems, protein mimics and building blocks for the formation of larger supra-molecular and super-molecular structures. At the present time, a large variety of dendrimers and sophisticated chemical modifications are known. However, the knowledge of their chemical preparation is not balanced by an equivalent understanding of the thermodynamic and kinetic properties of dendrimer systems. This understanding is important for the applications listed above.

Figure 2. Globular branched structure of a dendrimer (A) compared to the structure of a linear polymer (B).

I am interested in studying the thermodynamic, diffusion and phase transition properties of dendrimer solutions. Since dendrimers of different size exist and a large variety of functional groups can be easily attached on the dendrimer surface, dendrimer solutions can be systematically studied as a function of the dendrimer size and surface properties. These studies are not only relevant for the dendrimer systems themselves, but they can also be used to understand general properties of macromolecular systems. One of my goals is to study and control phase transitions that take place because macromolecules of different size are mixed together. This phenomenon is known as “macromolecular crowding”.

Drug Delivery. Drug delivery systems have the function to protect labile drugs and to transport them to targets remote from their administration sites. They also need to release the active agents in a predictable and controllable fashion. Thus, the development of drug delivery technologies greatly depends on biophysical, biochemical and physiological characteristics of the drug loaded devices. Drug delivery systems such as nano/microspheres are made of polysaccharides, proteins, or polymers. One aspect I intend to explore is the development of novel methods for the preparation of delivery systems. Several of the present devices for the delivery of drugs are made by elaborate preparations, usually involving phase separation methods. These techniques often employ organic solvents or high temperatures. These procedures have the potential to destroy the activity of labile drugs, such as proteins or polypeptides. Furthermore, residual amounts of solvents in the devices may pose significant health risks. I am interested in exploring new procedures for the preparation of drug delivery systems, which are based on phase transitions and self-assembly, and aim to avoid or at least minimize the use of elevated temperatures or toxic solvents.

Diffusion in Multicomponent Systems. Measurements of diffusion coefficients are typically used to predict and model the kinetics of several in vivo, laboratory, medical, pharmaceutical and manufacturing applications. Diffusion in multicomponent mixtures is usually approximated by defining one single diffusion coefficient for each solute, where the diffusion coefficient relates the concentration gradient of the solute to its flux (Fick’s law). This is however only an incomplete description. In fact, if a solution contains more than two components, the fluxes of the components are also coupled with each other. For instance, in the case of a ternary solution (two solutes and one solvent) a matrix of four diffusion coefficients is required: two main diffusion coefficients which describe the flux of the two solutes due to their own concentration gradients and two cross diffusion coefficients which describe the flux of each solute due to the gradient of the other solute. The determination of the diffusion coefficient matrix requires the use of a highly precise technique such as optical interferometry. Fig. 3 shows the interferometric pattern created by the diffusion mixing of two aqueous solutions with different solute concentrations.

Here at TCU we have developed a novel method that uses the diffusion coefficient matrix to determine the effect of an additive on the chemical potential of a macromolecule with high precision. This in turn provides information on the effective charge of the macromolecule, salting-out properties of the additive and macromolecular crowding. I intend to use this method in relation to both protein and dendrimer solutions.

Figure 3. Rayleigh interference fringes created by the diffusion mixing of two aqueous solutions.

I am also interested in using the concept of coupled diffusion to design novel pharmacokinetic profiles of drug release. In several systems the release of the drug occurs by diffusion of the drug through a microporous media. In these cases the flux of the drug at the surface of the delivery media and its time dependence are the factors to be controlled. Kinetic models for diffusion-mediated release of drug from delivery devices are derived from Fick's diffusion laws. An optimal drug release behavior requires a controllable release rate in many cases. The introduction of another diffusible component in the drug delivery device can act on the flux of the drug by coupled diffusion and thus regulate drug release profiles.

Selected Publications

O. Annunziata, O. Ogun and G. B. Benedek, “Observation of liquid-liquid phase separation for eye-lens gS-crystallin”, Proc. Natl. Acad. Sci. USA 100, 970-974 (2003).

O. Annunziata and J. G. Albright, “Mathematical model for diffusion of a protein and a precipitant about a growing protein crystal in microgravity”, Ann. NY Acad. Sci. 974, 610-616 (2002).

O. Annunziata, N. Asherie, A. Lomakin, J. Pande, O. Ogun and G. B. Benedek, “Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions”, Proc. Natl. Acad. Sci. USA 99, 14165-14170 (2002).

O. Annunziata, L. Paduano, A. J. Pearlstein, D. G. Miller and J. G. Albright, “Extraction of thermodynamic data from ternary diffusion coefficients.”, J. Am. Chem. Soc. 122, 5916-5928 (2000).

J. G. Albright, O. Annunziata, D. G. Miller, L. Paduano and A. J. Pearlstein, “Precision measurements of binary and multicomponent diffusion coefficients in protein solutions relevant to crystal growth”, J. Am. Chem. Soc. 121, 3256-3266 (1999).