Specific Aim 1: To determine if CpG HGNs induce direct apoptosis in lymphoma cell lines. HGNs are synthesized via a galvanic exchange reaction in which gold tetrachloroauric acid (HAuCl4) is reduced onto silver nanoparticles serving as sacrificial templates.15,16 We have extensive experience synthesizing complex HGNs that include theranostic features such as contrast agent for MRI or infrared luminance with quantum nanocrystals.17,18 The goal of this aim is to characterize the properties and efficacy of the CpG HGNs using in vitro lymphoma models. Sub-Aim 1.1: Particle characterization. Particle size is important for cellular endocytosis and gold shell thickness and size are important for tuning the absorbance to the near infrared region to maximize PTT efficacy. CpG per nanoparticle will be determined by mercaptoethanol exchange.9 Correlation between optical density with particle concentration and dose of CpG ODNs will be verified in this sub-aim. Sub-Aim 1.2: CpG HGN effects on lymphoma cell lines. Direct cytotoxic effects of CpG HGNs will be tested in various lymphoma cell lines in comparison to free CpG ODNs by viability assays. Cell lines include a double hit (MYC and BCL-2 re-arranged) DLBCL (RC), germinal center DLBCL (SUDHL4), activated B cell DLBCL (TMD8), Burkitt’s lymphoma (Ramos), and three murine lymphoma cell lines. We will evaluate changes in expression of OX40, PDL1 (as targets for checkpoint inhibitors), SR-B1 (targets for HDL mimic nanoparticles by our group), as well as maturation markers and cytokine releases. Sub-Aim1.3: Immune stimulatory effects of CpG HGNs on APCs. Murine macrophage cells and immature murine dendritic cells will be cultured with CpG HGNs and compared with free CpGs at various time points. Activation of the macrophages and dendritic cells will be monitored and quantified by evaluating the upregulation of maturation markers with flow cytometry. ELISAs will be used to quantify inflammatory cytokine release. Expected results and contingency plans: We expect that the particles will have higher cytotoxic effects on lymphoma cells. We expect increase in OX40 and increase in maturation markers, as previously published. For murine lymphoma lines, A20 is known to respond to CpGs and we anticipate that the nanoparticles will enhance the cytotoxic effects. CpG HGNs should mature antigen presenting cells and induce pro-inflammatory cytokine release. Specific Aim 2: To determine if systemic CpG HGNs delivery triggers anti-tumor immune response in immune competent lymphoma models. To fully understand the efficacy of CpG HGNs, an immune competent model is needed, such as the A20 lymphoma model. Sub-Aim 2.1: Toxicity and maximum tolerated dose of CpG HGNs. We will investigate the toxicity of CpG HGNs with ascending doses. The up-and-down testing method is most recommended by regulatory agencies for vertebrate animals.19 Initially, mice will receive 1011 particles/ml of CpG HGNs. The dose will be doubled and blood samples will be collected until any of the animals die, show signs of toxicity in blood work, or after 10 rounds of ascending doses. Mice would be sacrificed on day 15 and the liver, spleen, lung, and kidneys, will be fixed and sectioned for pathology review for toxic changes. Dose limiting toxicities will be compiled. Sub-Aim 2.2: Treatment of lymphoma using CpG HGN in a murine B cell lymphoma model. A20 lymphoma cells will be implanted on the flank of BALB/c mice as well as injected IV to mimic advanced stage disease. For the survival study, CpG HGNs will be injected via tail vein onc
|Effective start/end date||7/1/19 → 6/30/20|
- American Society of Hematology (Agmt 5/1/19)
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