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In Vivo Nano-Bio Interactions in Zebrafish using Advanced Fluorescence Microscopy
Monday, February 13, 2017 energy. This independent release has never been demonstrated before. This innovative delivery platform would be powerful medical and scientific tools to advance modern targeted biomolecule delivery technologies and resolve challenges for loading, delivery, and release of multiple drugs. 1487-Pos Board B555 Filovirus Mimics Deliver Effectively to Irreversibly Control Carcinoma Cell Fate Praful R. Nair, Cory Alvey, Dennis Discher. University of Pennsylvania, Philadelphia, PA, USA. Filoviruses are microns long, but biophysical advantages for such encapsulating/enveloped viruses are obscure. Flexible ‘filomicelles’ have been selfassembled from amphiphilic block copolymer (PEG-PCL) and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A, transcriptionally regulated by RA receptors, is a structural protein surrounding chromatin and can affect differentiation and karyokinesis. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis, which greatly increases cell death. When cancer cells are treated with either of the drugs alone, over several periods of the normal cell cycle, they revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual drug treatment with RA plus Paclitaxel increases aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects being durable. A month after treatment, relapse rates are low for RA-Paclitaxel treated cells (15%), compared to almost all (92%) for cells treated with Paclitaxel alone. Reduction in levels of key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for this synergy. These effects are greatly enhanced by loading the drugs into filomicelles. Co-loading the drugs into filomicelles lead to a more potent system compared to separate loading, with no loss in the integration efficiency of drugs. Notably, relapse rates were ~2% three months after treatment, highlighting the improvement offered by filomicelles. The combination retains its potency across multiple cell lines (liver, lung and bone) despite varying responsiveness to RA alone. Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. Injections of free RA-Paclitaxel combination are able to arrest growth of subcutaneous lung tumors for over 15 days, a feat not achieved by Paclitaxel alone. Drug loaded filomicelles are able to control the growth of subcutaneous lung and liver tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation. Flexible filomicelles circulate longer and accumulate better in the tumors, thus increasing the efficacy of delivery and treatment. 1488-Pos Board B556 In Vivo Nano-Bio Interactions in Zebrafish using Advanced Fluorescence Microscopy Marta d‘Amora1, Giuseppe Sancataldo1,2, Romuald Intartaglia1, Alberto Diaspro1,3. 1 Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2Dibris, University of Genoa, Genoa, Italy, 3Department of Physics, University of Genoa, Genoa, Italy. The huge production and the demand of engineered nanomaterials for biomedical applications give rise to an urgent need to assess their biological effects and toxic potential (1). Within this framework, it is crucial to develop rapid and direct methods that provide a deep understanding of the behaviour, interactions and transport of nanoparticles inside complex biological systems at whole animal level (from cells to tissues and organs). Herein, we propose to couple zebrafish as in vivo vertebrate organism and advanced fluorescence imaging techniques for evaluating the potential consequences induced by the exposure and the uptake of florescent emitting nanomaterials on the embryo development (2). This study outlines the macroscopic effects and alterations in zebrafish embryos and larvae by assessing different exposure periods. Moreover, advanced fluorescence imaging techniques, thanks to their 3D capabilities and fast speed, allow non-invasive bio-imaging of the nanoparticles internalization, transport and tissues accumulation during zebrafish development (3). Our results demonstrate the capability of this approach for exploring the nanoparticles bio-interactions in order to address the safe use of engineered nanomaterials suitable for clinical applications. (1) Mu Q. et al., Chemical Reviews; 114(15): 7740-7781 (2014) (2) d‘Amora M. et al., Scientific Reports; 6: 33923 (2016)(3) Lavagnino Z. et al., Scientific Reports; 6: 23923 (2016).
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1489-Pos Board B557 Half Pegylated Particles Evade Macrophages as Effectively as Fully Pegylated ones Lucero Sanchez, Yi Yi, Yan Yu. Chemistry, Indiana University, Bloomington, IN, USA. A grand challenge in developing therapeutic nanoparticles is to control their interaction with immune cells, such as macrophages. Nanoparticles in the blood stream are detected by immune cells as ‘‘foreign’’ due to the adsorption of blood serum proteins on the particle surface. This leads to the failure of most drug delivery particles. One strategy to overcome this challenge is to coat particles with polyethylene glycol (PEG) as a protective layer to prevent serum protein adsorption. Unfortunately, the bulky polymer chains also hinder the particle binding to their target cells. Therefore, new particle designs are necessary to circumvent this problem. In this presentation, I will report a method to decouple the bulky polymer chains and cell-specific ligands by creating twofaced Janus nanoparticles with different surface chemistries on opposing sides. Our results show that the half-PEGylated Janus nanoparticles are internalized by macrophages at a low probability, comparable to fully PEGylated nanoparticles. Meanwhile, the targeting side of the Janus particles binds cells unidirectionally, bypassing the hindrance from the PEGs on the other hemisphere. A new mechanism will be presented to explain how a spatial coating of PEGs on particles changes the phagocytosis of particles. This study suggests that the spatial decoupling of PEGs and cell-targeting moieties may be an effective strategy for designing therapeutic particles that evade immune detection and target cells without interference between the two functions. 1490-Pos Board B558 Atomic Force Microscopy as a Tool to Evaluate the Risk of Cardiovascular Diseases in Patients Ana F. Guedes1, Filomena Carvalho1, Nuno Lousada2, Luis Sargento2, Nuno C. Santos1. 1 Instituto de Medicina Molecular, Lisboa, Portugal, 2Unidade de Insuficieˆncia Cardı´aca, Departamento de Cardiologia, Hospital Pulido Valente, Lisboa, Portugal. High fibrinogen levels are a relevant cardiovascular risk factor, but the biological mechanisms associated with pathologic alterations are not totally clear. Fibrinogen-erythrocyte binding in chronic heart failure (CHF) patients and its prognostic value were evaluated. 15 ischemic and 15 non-ischemic CHF patients, as well as 15 healthy donors were enrolled in the study. Fibrinogen-erythrocyte interactions were evaluated, at the single-molecule level, by atomic force microscopy (AFM)-based force spectroscopy. Clinical outcome was assessed during a 12-months follow-up. AFM stiffness studies were also performed on erythrocytes from these patients and compared with the control. Force spectroscopy data showed that CHF patients presented higher fibrinogenerythrocyte binding forces than the control group, despite a lower binding frequency. According to etiology, ischemic patients had higher binding forces than donors and lower binding frequency. Ischemic patients presented increased fibrinogen-erythrocyte binding forces relative to non-ischemic. Non-ischemic patients also had a lower binding frequency than donors. Increased cell stiffness (or decreased elasticity) was determined for both groups of CHF patients, with the larger variation being observed for the non-ischemic patients. Clinical follow-up data demonstrated that patients presenting higher fibrinogen-erythrocyte binding forces at the beginning of the study had a higher probability of being hospitalized due to cardiovascular complications on the subsequent year. As fibrinogen-erythrocyte interactions, evaluated by AFM, are modified in CHF patients and associated with short-term clinical outcome, here we demonstrate the power of this biophysical evaluation as potential biomarker for cardiovascular risk and patients’ clinical prognosis evaluation [Guedes et al. (2016) Nature Nanotechnol., doi:10.1038/nnano.2016].
Bioengineering 1491-Pos Board B559 Computational Cell-Barcoding for High-Throughput Robotic Microscopy Mariya Barch1,2, Gaia Skibinski1,2, Alicia Lee1,2, Steven Finkbeiner1,3. 1 Gladstone Institute, San Francisco, CA, USA, 2Taube-Koret Center for Neurodegenerative Disease Research, San Francisco, CA, USA, 3 Departments of Neurology and Physiology, University of California, San Francisco, San Francisco, CA, USA. Studies of individual cells within populations rely on the ability to identify the same cell in multiple images. While high sampling frequencies in time-lapse movies greatly simplify the tasks of tracking cells spatially and stimulusresponses temporally, delicate samples undergoing longitudinal study might
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1489-Pos Board B557 Half Pegylated Particles Evade Macrophages as Effectively as Fully Pegylated ones Lucero Sanchez, Yi Yi, Yan Yu. Chemistry, Indiana University, Bloomington, IN, USA. A grand challenge in developing therapeutic nanoparticles is to control their interaction with immune cells, such as macrophages. Nanoparticles in the blood stream are detected by immune cells as ‘‘foreign’’ due to the adsorption of blood serum proteins on the particle surface. This leads to the failure of most drug delivery particles. One strategy to overcome this challenge is to coat particles with polyethylene glycol (PEG) as a protective layer to prevent serum protein adsorption. Unfortunately, the bulky polymer chains also hinder the particle binding to their target cells. Therefore, new particle designs are necessary to circumvent this problem. In this presentation, I will report a method to decouple the bulky polymer chains and cell-specific ligands by creating twofaced Janus nanoparticles with different surface chemistries on opposing sides. Our results show that the half-PEGylated Janus nanoparticles are internalized by macrophages at a low probability, comparable to fully PEGylated nanoparticles. Meanwhile, the targeting side of the Janus particles binds cells unidirectionally, bypassing the hindrance from the PEGs on the other hemisphere. A new mechanism will be presented to explain how a spatial coating of PEGs on particles changes the phagocytosis of particles. This study suggests that the spatial decoupling of PEGs and cell-targeting moieties may be an effective strategy for designing therapeutic particles that evade immune detection and target cells without interference between the two functions. 1490-Pos Board B558 Atomic Force Microscopy as a Tool to Evaluate the Risk of Cardiovascular Diseases in Patients Ana F. Guedes1, Filomena Carvalho1, Nuno Lousada2, Luis Sargento2, Nuno C. Santos1. 1 Instituto de Medicina Molecular, Lisboa, Portugal, 2Unidade de Insuficieˆncia Cardı´aca, Departamento de Cardiologia, Hospital Pulido Valente, Lisboa, Portugal. High fibrinogen levels are a relevant cardiovascular risk factor, but the biological mechanisms associated with pathologic alterations are not totally clear. Fibrinogen-erythrocyte binding in chronic heart failure (CHF) patients and its prognostic value were evaluated. 15 ischemic and 15 non-ischemic CHF patients, as well as 15 healthy donors were enrolled in the study. Fibrinogen-erythrocyte interactions were evaluated, at the single-molecule level, by atomic force microscopy (AFM)-based force spectroscopy. Clinical outcome was assessed during a 12-months follow-up. AFM stiffness studies were also performed on erythrocytes from these patients and compared with the control. Force spectroscopy data showed that CHF patients presented higher fibrinogenerythrocyte binding forces than the control group, despite a lower binding frequency. According to etiology, ischemic patients had higher binding forces than donors and lower binding frequency. Ischemic patients presented increased fibrinogen-erythrocyte binding forces relative to non-ischemic. Non-ischemic patients also had a lower binding frequency than donors. Increased cell stiffness (or decreased elasticity) was determined for both groups of CHF patients, with the larger variation being observed for the non-ischemic patients. Clinical follow-up data demonstrated that patients presenting higher fibrinogen-erythrocyte binding forces at the beginning of the study had a higher probability of being hospitalized due to cardiovascular complications on the subsequent year. As fibrinogen-erythrocyte interactions, evaluated by AFM, are modified in CHF patients and associated with short-term clinical outcome, here we demonstrate the power of this biophysical evaluation as potential biomarker for cardiovascular risk and patients’ clinical prognosis evaluation [Guedes et al. (2016) Nature Nanotechnol., doi:10.1038/nnano.2016].
Bioengineering 1491-Pos Board B559 Computational Cell-Barcoding for High-Throughput Robotic Microscopy Mariya Barch1,2, Gaia Skibinski1,2, Alicia Lee1,2, Steven Finkbeiner1,3. 1 Gladstone Institute, San Francisco, CA, USA, 2Taube-Koret Center for Neurodegenerative Disease Research, San Francisco, CA, USA, 3 Departments of Neurology and Physiology, University of California, San Francisco, San Francisco, CA, USA. Studies of individual cells within populations rely on the ability to identify the same cell in multiple images. While high sampling frequencies in time-lapse movies greatly simplify the tasks of tracking cells spatially and stimulusresponses temporally, delicate samples undergoing longitudinal study might