UCLA

Nanomedicine focuses on the development and engineering of novel and unique therapeutic and diagnostic agents that can overcome the challenges associated with using traditional modalities. Nanoparticles (NPs) in the size range between 1 and 1000 nm have many advantages for use in these applications, such as, low polydispersity, established characterization methodologies, and the ability to be loaded with therapeutics for diseases, conjugated to targeting ligands to enhance specificity, and coated with polymers to improve stability and half-lives.

This thesis focused on developing nanotechnology to address both therapeutic and diagnostic applications due to the universal need to understand and maintain colloidal stability. Maintaining colloidal stability is crucial in therapeutic applications since aggregated NPs will have a reduction in the number of targeting ligands available for interacting with cell-surface receptors by reduction of the surface area to mass (or volume) ratio. The drug release from the NPs can also be significantly altered, and the aggregate sizes may no longer be appropriate for targeting tumors passively and for being actively internalized into cancer cells. The aggregation also has the possibility of severely reducing the shelf-life of the drug carriers. In the case of diagnostics, in addition to reducing the number of targeting ligands and shelf-life, aggregation can change the optical properties of NPs. Moreover, aggregated NPs in the well-established lateral-flow immunoassay (LFA) can experience difficulty flowing through the test strip and may give rise to invalid results. Accordingly, irrespective of the application, the studies in this thesis required an understanding and tuning of the electrostatic and steric, excluded-volume stabilizing interactions when designing the NPs.

With regard to cancer therapy, this thesis focused on further testing the limits of a transferrin (Tf) variant that was previously engineered by our laboratory for improving the delivery of cytotoxins in a Tf-cytotoxin molecular conjugate format. Specifically, our previously derived mathematical model was first extended to theoretically determine if conjugating a Tf variant to a NP would increase its association with a cancer cell, thereby increasing the probability of delivering a toxic payload to a cancer cell. Upon finding that the theoretical predictions supported such a construct, the Tf variant was conjugated to the NPs and then radiolabeled with iodine-125, and these NPs were found to associate with prostate cancer cells for a greater period of time relative to the native Tf counterpart, as predicted by our extended mathematical model. Subsequently, poly(lactide-co-glycolide) (PLGA) NPs encapsulating the chemotherapeutic doxorubicin (DOX) was conjugated to a Tf variant and was shown to have improved drug carrier efficacy both in vitro and in vivo relative to the native Tf counterpart. The in vivo results were especially exciting as a single intravenous injection was found to dramatically inhibit tumor growth in a mouse model for prostate cancer. This work corresponded to the first ever investigation of the drug carrier properties of NPs conjugated to a Tf variant. In contrast to our laboratory's previous studies with molecular drug conjugates, which are very promising for cancers treated locally, these Tf variant-conjugated NPs administered intravenously have potential for treating most types of cancers.

With regard to diagnostic applications, this thesis focused on revolutionizing LFA. One of the most common applications of LFA is the pregnancy test, where gold NPs are used as the colorimetric indicator. The small sizes of the gold NPs also allow the use of low sample volumes, and their large surface area to mass ratio enables efficient capture of target molecules. Their optical properties can also be exploited to directly visualize a result. Although LFA has many features that make it attractive for use as a point-of-care diagnostic, the use of LFA for detecting biomarkers at low concentrations is severely limited due to its low sensitivity. To increase LFA sensitivity, our laboratory is the only one to have used aqueous two-phase complex fluid systems for concentrating biomarkers prior to their detection. This pioneering work is described in the thesis, where we began by using an aqueous two-phase Triton X-114 micellar system to pre-concentrate the model virus M13 prior to its detection with LFA.

This first combination of an aqueous two-phase system (ATPS) and LFA improved the detection limit of LFA for M13 by 10-fold, and has led to many new exciting avenues of research. Subsequently, an aqueous two-phase polyethylene glycol (PEG)-salt system was investigated to concentrate the same virus but in a much shorter time period. The same PEG-salt system was then examined for its ability to concentrate the model protein Tf. In this case, due to Tf being significantly smaller than M13, gold NPs were used to bind and transport Tf into one of the two phases of the aqueous two-phase PEG-salt system. However, due to the high concentrations of salt present in one of the phases, the gold NPs were manipulated to ensure colloidal stability through the entire process from concentration to detection. These gold NPs were also manipulated to drive the gold NPs to the interface instead of one of the two bulk phases with the idea of further concentrating the target biomolecules, as the interface corresponds to a three-dimensional region that is only a few molecular diameters thick. Moreover, this thesis discusses the discovery of a very exciting phenomenon where the paper membranes used in LFA can enhance the phase separation process of an ATPS, which enables the concentration of the target molecules as they flow directly on the test strip. This ability to simultaneously concentrate and detect biomarkers directly on paper removes the need to extract a phase from a test tube and also reduces the time to result. Lastly, the combination of an ATPS and LFA was further extended to include magnetic fields and a new solid-liquid interface to enhance extraction. All of these technologies have the potential to dramatically improve the state of health care in resource-poor settings by providing rapid, accurate, and inexpensive diagnostics, leading to improved patient management, treatment, and outbreak prevention.