Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmacology and Experimental Neuroscience

First Advisor

Dr. Howard Gendelman

Abstract

RATIONALE: Our laboratories birthed the field of human immunodeficiency virus (HIV) theranostics. The new field allows simultaneous detection (diagnostics) and treatment (therapeutic) for the identification, treatment and inevitable elimination of virus in cell and tissue compartments. By employing theranostics, antiretroviral drugs (ARVs) can be tracked in lymph nodes, gut, spleen and liver. Cellular viral reservoirs including CD4+ T cell populations and mononuclear phagocytes (MP; monocytes, macrophages, microglia and dendritic cells) along with subcellular endosomal structures can now be targeted for drug delivery bringing therapeutics to areas where virus replicates. The overarching idea rests in improving precision targeted ARV delivery. Bringing ART to anatomically privileged tissues can be visualized and confirmed through single photon emission computed tomography (SPECT) imaging facilitated by multimodal antiretroviral drug (ARV) nanoprobes. To deploy such technologies, we have successfully placed rilpivirine into a theranostic nano system, taking advantage of state of the art physical and chemical properties of nano-sized particles for maximal biodistribution to viral sites. This allowed measurements of optimal antiretroviral responses. To achieve this outcome, we made particles with combinations of bioimaging detectors and ARV deliverers. This platform, in future studies, will utilize LASER ART and HIV-1 excision payloads (for example, CRISPR Cas9) for the inevitable elimination of viral infection. Our overall goal is to facilitate long-acting slow effective release antiretroviral therapy (LASER ART) development. To this end, in a first step analysis we created “multimodal imaging theranostic nanoprobes” with the hydrophobic antiretroviral drug rilpivirine (RPV). These unique nanoprobes allowed combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. Combination of SPECT/CT and MR imaging modalities resulted in a highly accurate and sensitive nanoprobe. Because of this combination, the imaging data acquired from these nanoprobes after administration in mice proved predictive of future drug pharmacokinetics and biodistribution.

METHODS: 111Indium (111In) and Europium (Eu3+)-doped cobalt ferrite (CF) rilpivirine (RPV)-loaded (111InEuCF-RPV) nanoparticles were synthesized then fully characterized based on their size, shape and stability. The particles were tested in vitro for uptake, retention and antiretroviral efficacy in human monocyte-derived macrophages (MDMs) along with the intracellular location of particles. These were then used as platforms for nanoformulated drug biodistribution. For multimodal imaging and biodistribution studies; 111InEuCF-RPV, ultra-small lipid coated 177LuEuCF and NRPV particles were injected intravenously into mice at various concentrations of drug or radioisotope. One group was treated with ultra-small lipid-coated 177LuEuCF particles at ∼ 74 MBq (2000 μCi) to assess the effect of particle size on biodistribution. Drug levels were quantified in plasma and tissues by UPLC-MS/MS and cobalt levels were quantified by ICP-MS. Pearson’s correlations were used to assess the predicative potential of imaging data and future ARV biodistribution and pharmacokinetics.

RESULTS: 111InEuCF-RPV particles were synthesized and were shown to be of consistent size and were stable in a variety of different media conditions for over a week. Physiochemical characterizations and TEM imaging confirmed the structure and components of the system were correct. Formed theranostic particles were shown to be non-toxic to MDMs and at high concentrations were much less cytotoxic than native RPV. Particles demonstrated excellent intracellular relaxivity values of r2 = 732.8 mM-1s-1 and thus served as excellent MRI contrast agents. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses similar to nanoformulated RPV. After administration to Balb/c mice particles could be localized to the spleen, liver, as well as popliteal and axillary lymph nodes. Imaging showed that nanoparticles accumulated in the spleen over 5 days and gradually left the liver as confirmed by ex vivo autoradiographic imaging and gamma scintillation spectrometry. Imaging data acquired up to 5 days proved predicative of drug biodistribution and pharmacokinetics up to 28 days post administration.

CONCLUSIONS: We conclude that this novel nano system can be used broadly for theranostic antiretroviral drug biodistribution. In particular, in the not so distant future, it will enable the merger of LASER ART with detection methods to realize the long term goal of improving patient outcomes by assessing where and to what levels antiretroviral drugs are delivered into viral compartments. The long term goals are to best prevent new infections, achieve viral elimination, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs.

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