UC Berkeley

The following dissertation presents work done towards the development of delivery strategies for proteins and nucleic acids to mammalian cells, as well as targeted imaging materials to bacteria. Delivery of large molecules such as proteins, nucleic acids, and large synthetic molecules remains a major bottleneck in the field of drug delivery, and despite an abundance of research into the topic, the delivery of intracellular proteins has still not seen clinical significance. To address this, we sought to develop new delivery vehicles in an endeavor to address current challenges in drug delivery.

Chapter 1 discusses the development of acid-sensitive surfactants for the delivery of nucleic acids. This class of molecules, termed caged surfactants, consists of a membrane disruptive surfactant that has been masked with two PEG chains through an acetal linker. This prevents it from entering the membrane in its intact state; however, after acetal hydrolysis in the acidic endosomal compartment, release of free surfactant causes endosomal release of any co-delivered cargo. The simultaneous delivery of caged surfactant with nucleic acid was achieved using two strategies: (i) inclusion of two primary amines on the caged surfactant to allow complexation through electrostatic interactions, and (ii) inclusion of the RNA-binding dye thiazole orange (TO) to allow tight binding through intercalation. In this study we show that amine-containing caged surfactant (PCS) and TO-containing caged surfactant (TCS) increase the delivery of mRNA and siRNA, respectively.

Chapter 2 discusses the development of delivery vehicles for the delivery of proteins to mammalian cells. The chapter consists of two separate studies. The first study utilized a fluorescent acid-sensitive membrane disruptor (FEDS) as an adjuvant to Lipofectamine-based delivery of CRISPR-Cas9. FEDS successfully increased the delivery efficacy of Cas9 to HEK cells. In addition, due to its fluorescent properties the cells with Cas9-triggered gene editing could be sorted through a fluorescence cell sorter. The second study describes the development of a polyethylene glycol-Eosin Y block copolymer (PEG-pEosin) for protein delivery. The Eosin Y moieties bind a wide range of proteins, and PEG-pEosin can therefore be used as a general protein delivery tool. Using PEG-pEosin we successfully delivered Cre recombinase to Ai9 cells through a PEG-pEosin/Cre/Listeriolysin O complex, wherein the Lysteriolysin O serves as an endosomal disruptive component.

Chapter 3 presents a maltohexaose-indocyanine green conjugate for the detection of infective endocarditis. Maltohexaose (MH) is a sugar that is specifically taken up by bacteria, and it is relatively tolerant of modifications to its reducing end. The FDA-approved dye indocyanine green (ICG) was conjugated to MH to yield a low toxicity, high wavelength probe that was shown to accumulate in bacterial vegetations in an endocarditis rat model.