Encapsulation and controlled release of active DNA from uncrosslinked gelatin microspheres

This thesis work investigates the encapsulation of DNA in gelatin microspheres (GMS) and the subsequent temperature controlled release of the encapsulated DNA from these GMS. DNA-loaded GMS were then used as templates for colloidal satellite assemblies and the released DNA was shown to competitively displace the original partner strands of immobilized DNA on the surface of the assemblies. To support these investigations, hybridization of DNA at colloidal surfaces was also investigated using in situ measurements. DNA hybridization is of particular interest as means of controlling the functionality of colloidal structures because it is uniquely reversible and tunable as well as biocompatible. Gelatin was chosen as the encapsulation matrix for its superior biocompatibility, convenient gel to liquid phase transition at ∼35°C, and economical availability.;This thesis is divided into five chapters. Chapter 1 covers the motivation of this work and provides a general background for the materials used. Chapter 2 details the synthesis of GMS and the use of these uncrosslinked GMS as controlled release matrices for active DNA. Bare GMS were not found to be able to inhibit DNA release on their own. With the addition of a polyelectrolyte bilayer, however, clear inhibition of DNA release at room temperature and permitted release at 37 °C was observed. Chapter 3 is an investigation of the thermodynamics and kinetics of primary and secondary DNA hybridization at colloidal surfaces. Flow cytometry was used to quantify the hybridization reaction in situ and compare it to more conventional measurement protocols involving washing steps. The post washing results illuminated the importance of the toehold region and demonstrated changes in kinetics with changing toehold length which are consistent with published solution studies of toehold-mediated strand displacement. The in situ studies enabled the measurement of primary hybridization rate as well as secondary hybridization rate. Despite the significant deviation in degree of hybridization that washing steps can induce, the in situ and post washing results were still similar in their overall trends. Chapter 4 details the use of microfluidics to manufacture monodisperese GMS as well as the subsequent assembly of colloidal satellite structures using these GMS. DNA released from the GMS template particle was found to competitively displace fluorescently labeled primary hybridization partners on the DNA-functionalized satellite particles, thus changing the duplex “expression” of the surface of the colloidal assembly. Chapter 5 offers some concluding remarks and some areas for future exploration.