Exploring the formation of guanosine-based colloidal particles and their affinity towards targeted molecules

Abstract

The ultimate goal of stimuli-responsive drug delivery systems is the encapsulation and release of biologically relevant molecules (e.g., drugs, proteins) with spatiotemporal control. In order to do this, delivery systems are studied in a laboratory setting to fine-tune the particle formation and encapsulation/release properties. The encapsulation of targeted molecules can be accomplished via several methods including physical entrapment or absorption of the guest within the particle. Affinity-based drug delivery systems use non-covalent interactions as the driving force behind the incorporation and delivery of desired guests. Furthermore, the quantification of the encapsulation/release data of these systems often relies on methods that require sample manipulation (e.g., centrifugation and measurement of supernatant) that is not suitable for some materials. With this in mind, in Chapter 2 we present the development of a new method, based on Flow Cytometry, for determining the affinity of small molecules in colloidal particles made from guanosine assemblies. These hierarchically organized colloidal particles developed by us are termed supramolecular hacky sacks (SHS). These SHS particles, made via the self-assembly of guanosine (G)-derivatives, are suitable for the encapsulation of small molecules like fluorescent dyes and biomacromolecules like proteins and oligonucleotides. We use Flow Cytometry and Confocal Laser Scanning Microscopy to quantify the change in fluorescence of the SHS particle before and after the encapsulation of a variety of fluorescent dyes at various concentrations. The results show that molecules with a positive charge (e.g., thiazole orange) had the highest affinity among the compounds tested. In Chapter 3, we use apply these methods to measure the affinity of fluorescently labeled proteins towards the SHS particles. The results show that proteins have moderate affinity towards high molecule weight guests like proteins. Knowing the baseline affinity for prototypical proteins, we demonstrate a proof-of-concept study showing the possibility of modulating the affinity of SHS particles via the incorporation of a biotin moiety as an affinity ligand to the guanosine derivative via aldehyde/hydrazide conjugation. Modification of the 2’-deoxyguanosine monomer encodes the necessary information to the resulting supramolecular structure to respond to biologically relevant stimuli such as hydrogen peroxide (H₂O₂) and enzymes. In Chapter 4 we show the development of a H₂O₂-responsive material via the encapsulation of a fluorescent probe in an SHS particle. In vitro studies with RAW 264.7 cells show that the particle responds to changes in concentration of H₂O₂, evidenced by an increase in fluorescence of the particles, as well as release of the encapsulated cargo. Chapter 5 describes initial studies towards the development of an enzyme-responsive supramolecule. Specifically, we show preliminary data about the formation of SHS particles by interaction with Alkaline Phosphatase. We also show multiple synthetic strategies towards G-derivatives that could in principle self-assemble into an enzyme-responsive supramolecule. In conclusion, this thesis describes our studies towards the control of particle formation and encapsulation/release properties of guanosine-based colloidal particles.