Browsing by Subject "Nanoparticles"
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Item The Design, Synthesis, and Evaluation of Zwitterionic and Cationic Lipids for In Vivo RNA Delivery and Non-Viral CRISPR/Cas Gene Editing(2018-03-05) Miller, Jason Brian; Ready, Joseph M.; Gao, Jinming; Tambar, Uttam; Siegwart, Daniel J.The delivery of nucleic acids is an emerging therapeutic modality in clinical development for the treatment of many genetic diseases. The use of RNA interference (RNAi) as a therapeutic is an exciting and rapidly developing field that offers a promising alternative to small molecule drugs for the treatment of dysregulatory diseases, including cancer. Small interfering RNA (siRNA) can be designed against any mRNA target, and upon loading into the RNA-induced silencing complex (RISC) can enable sequence-specific target recognition and degradation. Meanwhile, messenger RNA is currently being utilized for protein replacement therapy and for the development of vaccines by expressing viral antigens on dendritic cells. However, because RNA molecules are unable to passively diffuse across plasma membranes due to a high molecular weight (~13 kDa for siRNA, >300 kDa for mRNA), hydrophilicity and strong anionic charge, while also being unstable and highly immunogenic when injected systemically, nucleic acid therapeutics require carriers for effective delivery. To date, many successful carriers have been designed using amphiphilic lipid-like compounds containing amine-rich cores, but the challenges of efficient endosomal release and delivery to organs outside of the liver remain major hurdles in the field of RNA therapeutics. This dissertation reports the design, synthesis and characterization of two new classes of lipids with unique chemical structures and in vivo RNA delivery capabilities to the lung: zwitterionic amino lipids (ZALs) and cationic sulfonamide amino lipids (CSALs). ZALs contain an amine rich core, hydrophobic tails introduced via conjugate addition or epoxide opening, and a zwitterionic sulfobetaine head group. ZALs were designed with a combination of cationic and zwitterionic lipid properties, to help stabilize and effectively deliver long RNA molecules. A lead compound, ZA3-Ep10, was effective for in vivo messenger RNA delivery and the first reported demonstration of in vivo non-viral gene editing by delivering mRNA components encoding the CRISPR/Cas gene editing platform. CSALs contain a unique chemical scaffold containing an internal quaternary ammonium group and a sulfonamide linker. A rational investigation of structure-activity relationships revealed that CSALs containing an acetate sidearm, a dimethyl amino head group and higher hydrophobic content were effective in delivery siRNA to human cancer cells in vitro. CSALs also demonstrated lung localization upon systemic delivery in vivo while also demonstrating the ability to redirect liver targeting ionizable lipid nanoparticles to the lung. These new classes of materials demonstrate the importance of structural consideration in material design for the development of nucleic acid therapeutics, while also providing structural templates for developing carriers for effective delivery to tissues outside of the liver.Item Development of Iron Oxide Based Nanoparticles as Dual-Modality Imaging Probes(2008-09-12) Guo, Yi; Sun, XiankaiDual-modality (MR/nuclear) imaging can combine exquisite anatomical resolution with superior molecular sensitivity, and significantly facilitate the accuracy of cancer diagnosis. However, the application of this technique is hampered by the paucity of sensitive dual-modality imaging probes that target tumors specifically. Here we synthesized dual-modality imaging probes by doping positron- or gamma-emitting nuclides to the core of dextran-coated superparamagnetic iron oxide nanoparticles (NUSPIONs). The synthesized nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), atomic force microscope (AFM), and high performance liquid chromatography (HPLC). The evaluations of these nanoparticles were performed both in vitro and in vivo. Four radioisotopes (111In, 177Lu, 64Cu, and 77As) were successfully incorporated into the core of nanoparticles. The purification of nanoparticles via centricon filter accelerated the separation process effectively without apparent aggregation. These nanoparticles exhibited good in vitro stability in both phosphate buffered saline (> 99% intact) and rat serum (> 92% intact) out to 72 h, and the high r2-to-r1 ratio indicating their potential as MRI T2 contrast agents. Two distinctly sized 177Lu-doped nanoparticles (NUSPION-1 and NUSPION-2 with hydrodynamic radii of 11.8 ��3 nm and 30.6 ��5 nm respectively) were used for biodistribution studies in normal mice. NUSPION-1 showed significantly (p < 0.0001) higher uptake and longer retention in blood and less uptake in liver and spleen than NUSPION-2, which is advantageous for both passive and active targeting. Due to its optimal tissue distribution pattern, NUSPION-1 was chosen for further in vivo evaluation in PC-3 tumor-bearing mice. High tumor uptake and contrast ratios of tumor-to-muscle and tumor-to-blood were observed. A proof-of-principle dual-modality imaging study was carried out by a virtually single-dose injection in PC-3 tumor-bearing mice. The tumors were visualized by both MRI and autoradiography. Post-MRI Prussian blue iron staining and post-autoradiographic imaging biodistribution confirmed the accumulation of nanoparticles in tumors. Taken together, we have demonstrated a practical method to develop iron oxide based MRI/nuclear imaging probes.Item Illuminating Endocytic Organelles with pH-Resposive [sic] Nanomaterials(2017-02-20) Wang, Chensu; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming; Danuser, Gaudenz; Yoo, Hyuntae; Zhong, QingEndosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of key cell physiological processes that include protein/lipid metabolism, nutrient sensing and cell survival. To support quantitative investigation of these processes in living cells, we have developed a library of ultra-pH sensitive (UPS) fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatial-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution. Deployment in cells enabled quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. These biological applications benchmarked the robustness and adaptability of this nanotechnology-enabled 'detect and perturb' strategy. As a translational application, we leveraged the technology in high-throughput screening assays that successfully identified chemical agents in the promotion of autophagolysosomal activity through TFEB activation. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles conferred hepatoprotection against diet-induced steatosis in murine models and prolonged survival in Caenorhabditis elegans. These results highlight the therapeutic potential of small-molecule TFEB activators to ameliorate metabolic syndrome and extend lifespan.Item The Impact of Lipid Nanoparticle Chemistry on RNA Delivery and Therapeutic Outcomes(2022-12-01T06:00:00.000Z) Johnson, Lindsay Taylor; de Gracia Lux, Caroline; Siegwart, Daniel J.; Zhu, Hao; Hoshida, Yujin; Singal, Amit G.This dissertation aims to understand how two individual components of the traditional four-component lipid nanoparticle system, the PEG lipid component and the ionizable cationic lipid component, impact RNA delivery. To systematically investigate how PEG lipid chemistry impacted LNP formulation and RNA delivery, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids were synthesized with modulated hydrophobic domains. The chemical structure of the hydrophobic domain did not impact the formulation of 5A2-SC8 LNPs, including nanoparticle size, RNA encapsulation, and stability. However, the chemical structure did affect RNA delivery efficacy both in vitro and in vivo. The chemical structure of the hydrophobic domain of the PEG lipids impacted the escape of 5A2-SC8 LNPs from endosomes at early cell incubation time points. Overall, the results indicated that PEG lipid anchoring and chemical structure modulated RNA delivery. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), it was observed that two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) resulted in distinct RNA delivery within the liver organ. Despite both LNPs possessing similar physical properties, the ability to silence RNA in vitro, strong accumulation within the liver, and sharing a pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Protein corona analysis indicated that 5A2-SC8 LNPs bind Apolipoprotein E (ApoE), which can drive LDL-R receptor mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in Albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival compared the non-treatment group. Since disease targets exist in an organ- and cell-type specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. Overall, the results from this work illustrates the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.