Bio

Bio


Lab Manager, Stanford University, Prof. Tarik Massod08/2015 - Present
School of Medicine, Department of radiology section of Neuroradiology,
Molecular imaging program at Stanford (MIPS) Lab
• Performed antioxidant project to treat brain/breast cancer utilizing the following techniques :
-Cell culturing (T98G, LN229, U87, T98G, 293T, MDA, Hela cells) including maintenance of cells, transfection, transduction and drug treatment
-Cloning, transformation, plasmid extraction, gel electrophoresis
-Flow cytometer, mitochondria staining, luciferase assay
-Nanomedicine, Molecular imaging, Targeted delivery, Gene therapy
•Enhanced the therapeutic efficacy of Temozolomide (clinical drug) towards glioblastoma by 20 with miR-21 downregulation and PLGA nanoparticles
•Co-delivery of therapeutic microRNAs and drugs for triple negative breast cancer treatment in pre-clinical small animal model by multifunctional polymer nanocarriers
Canary Crest Summer Program Mentor, Stanford University, School of Medicine2017 & 2018
Assistant to the chair, Spiritual Care, UC Davis Med Center 10/2012-8/2015
Research Assistant, Stanford University, Prof. Ahmad Salehi10/2013 – 06/2015
•BDNF signaling in mouse model of Down syndrome:
Performed biochemical techniques including western blots, immunohistochemistry, qPCR and ELISA
•Study differences between normal and abnormal physiology to find appropriate biological targets for drug intervention
•Study the pathways by which the drugs are causing the observed effects on cognitive function and neurodegeneration
•Performed in-vitro and in-vivo experiments using mouse models of Down syndrome to study the effects of drugs on neurodegeneration and learning ability (cognition)
Research Assistant, University of California Davis, Prof. Kit Lam 6/2012-12/2012
Performed experiments on cancer treatment techniques including:
Library design, General library screening techniques, Optimizing concentration of dye
Image J, picked up beads, sequencing

Publications

All Publications


  • Intranasal delivery of targeted polyfunctional gold-iron oxide nanoparticles loaded with therapeutic microRNAs for combined theranostic multimodality imaging and presensitization of glioblastoma to temozolomide. Biomaterials Sukumar, U. K., Bose, R. J., Malhotra, M., Babikir, H. A., Afjei, R., Robinson, E., Zeng, Y., Chang, E., Habte, F., Sinclair, R., Gambhir, S. S., Massoud, T. F., Paulmurugan, R. 2019; 218: 119342

    Abstract

    The prognosis for glioblastoma (GBM) remains depressingly low. The biological barriers of the brain present a major challenge to achieving adequate drug concentrations for GBM therapy. To address this, we explore the potential of the nose-to-brain direct transport pathway to bypass the blood-brain barrier, and to enable targeted delivery of theranostic polyfunctional gold-iron oxide nanoparticles (polyGIONs) surface loaded with therapeutic miRNAs (miR-100 and antimiR-21) to GBMs in mice. These nanoformulations would thus allow presensitization of GBM cells to the systemically delivered chemotherapy drug temozolomide (TMZ), as well as in vivo multimodality molecular and anatomic imaging of nanoparticle delivery, trafficking, and treatment effects. First, we synthesized GIONs coated with beta-cyclodextrin-chitosan (CD-CS) hybrid polymer, and co-loaded with miR-100 and antimiR-21. Then we decorated their surface with PEG-T7 peptide using CD-adamantane host-guest chemistry. The resultant polyGIONs showed efficient miRNA loading with enhanced serum stability. We characterized them for particle size, PDI, polymer functionalization, charge and release using dynamic light scattering analysis, TEM and qRT-PCR. For in vivo intranasal delivery, we used U87-MG GBM cell-derived orthotopic xenograft models in mice. Intranasal delivery resulted in efficient accumulation of Cy5-miRNAs in mice treated with T7-targeted polyGIONs, as demonstrated by in vivo optical fluorescence and MR imaging. We measured the therapeutic response of these FLUC-EGFP labelled U87-MG GBMs using bioluminescence imaging. Overall, there was a significant increase in survival of mice co-treated with T7-polyGIONs loaded with miR-100/antimiR-21 plus systemic TMZ, compared to the untreated control group, or the animals receiving non-targeted polyGIONs-miR-100/antimiR-21, or TMZ alone. Once translated clinically, this novel theranostic nanoformulation and its associated intranasal delivery strategy will have a strong potential to potentiate the effects of TMZ treatment in GBM patients.

    View details for DOI 10.1016/j.biomaterials.2019.119342

    View details for PubMedID 31326657

  • Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo THERANOSTICS Nishihara, R., Paulmurugan, R., Nakajima, T., Yamamoto, E., Natarajan, A., Afjei, R., Hiruta, Y., Iwasawa, N., Nishiyama, S., Citterio, D., Sato, M., Kim, S., Suzuki, K. 2019; 9 (9): 2646–61

    View details for DOI 10.7150/thno.32219

    View details for Web of Science ID 000465285400015

  • Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo. Theranostics Nishihara, R., Paulmurugan, R., Nakajima, T., Yamamoto, E., Natarajan, A., Afjei, R., Hiruta, Y., Iwasawa, N., Nishiyama, S., Citterio, D., Sato, M., Kim, S. B., Suzuki, K. 2019; 9 (9): 2646–61

    Abstract

    Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named "Bottle Blue (BBlue)" and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

    View details for DOI 10.7150/thno.32219

    View details for PubMedID 31131059

    View details for PubMedCentralID PMC6525985

  • Comparison of cell-based assays to quantify treatment effects of anticancer drugs identifies a new application for Bodipy-L-cystine to measure apoptosis. Scientific reports Kumar, N., Afjei, R., Massoud, T. F., Paulmurugan, R. 2018; 8 (1): 16363

    Abstract

    Cell-based assays that measure anticancer drug effects are essential for evaluating chemotherapeutic agents. Many assays targeting various cellular mechanisms are available, leading to inconsistent results when using different techniques. We critically compared six common assays, as well as a new assay using Bodipy.FL.L-cystine (BFC), to identify the most accurate and reproducible in measuring anticancer drug effects. We tested three common chemotherapies (methotrexate, paclitaxel, and etoposide) in two cell lines (Ln229 and MDA-MB231). Spectroscopic assays such as Cell Titer Blue, and 2',7'-dichlorofluorescin diacetate (DCFDA) yielded a strong drug dose response, especially for paclitaxel and etoposide (R2=0.9). MTT and Calcein-AM fluorescent dye-based assays were less consistent in that regard. Among three flow cytometry assays, Propidium Iodide (PI)-based DNA content analysis and a new BFC-based glutathione-redox (GSH) assay produced drug dose dependent results. Compared to PI, BFC showed a better correlation (R2=0.7-0.9) in depicting live and apoptotic cells. We found that the combination of Cell Titer Blue spectroscopy and BFC flow cytometry assays were most accurate in assessing anticancer drug effects by clear distinction between live and apoptotic cells, independent of drug mechanism of action. We present a new application of BFC as an agent for measuring cellular apoptosis.

    View details for PubMedID 30397244

  • A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells. Oncotarget Paulmurugan, R., Afjei, R., Sekar, T. V., Babikir, H. A., Massoud, T. F. 2018; 9 (30): 21495–511

    Abstract

    Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.

    View details for PubMedID 29765555

  • Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model. Oncotarget Malhotra, M., Sekar, T. V., Ananta, J. S., Devulapally, R., Afjei, R., Babikir, H. A., Paulmurugan, R., Massoud, T. F. 2018; 9 (30): 21478–94

    Abstract

    Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.

    View details for PubMedID 29765554

  • Restoring guardianship of the genome: Anticancer drug strategies to reverse oncogenic mutant p53 misfolding. Cancer treatment reviews Babikir, H. A., Afjei, R., Paulmurugan, R., Massoud, T. F. 2018; 71: 19–31

    Abstract

    p53 is a transcription factor that activates numerous genes involved in essential maintenance of genetic stability. P53 is the most frequently mutated gene in human cancer. One third of these mutations are structural, resulting in mutant p53 with a disrupted protein conformation. Here we review current progress in a relatively underexplored aspect of p53-targeted drug development, that is, strategies to reactivate wild-type function of misfolded mutant p53. Unfortunately, most p53-targeted drugs are still at early stages of development and many of them are progressing slowly toward clinical implementation. Significant challenges need to be addressed before clinical translation of new anti-misfolding p53-targeted drugs.

    View details for PubMedID 30336366

  • Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents. ACS nano Jc Bose, R., Uday Kumar, S., Zeng, Y., Afjei, R., Robinson, E., Lau, K., Bermudez, A., Habte, F., Pitteri, S. J., Sinclair, R., Willmann, J. K., Massoud, T. F., Gambhir, S. S., Paulmurugan, R. 2018

    Abstract

    MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold-iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.

    View details for PubMedID 30346694

  • Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications. ACS chemical biology Gaur, S., Bhargava-Shah, A., Hori, S., Afjei, R., Sekar, T. V., Gambhir, S. S., Massoud, T. F., Paulmurugan, R. 2017

    Abstract

    Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.

    View details for PubMedID 28767220