Dhananjay Suresh

Bombesin Peptide Conjugated Gold Nanocages Internalize via Clathrin Mediated Endocytosis

The nature of interaction and mechanism of internalization of receptor-avid peptide nanoparticles with cells is not yet completely understood. This article describes the cellular internalization mechanism and intracellular trafficking of peptide conjugated receptor targeted porous Gold nanocages (AuNCs) in cancer cells. We synthesized and characterized a library of AuNCs conjugated with bombesin (BBN) peptide. Evidence of selective affinity of AuNC-BBN toward gastrin releasing peptide receptors (GRPR) was obtained using radiolabeled competitive cell binding assay. Endocytic mechanism was investigated using cell inhibitor studies and monitored using optical and transmission electron microscopy (TEM). Results show AuNC-BBN uptake in PC3 cells is mediated by clathrin mediated endocytosis (CME). Indeed, in the presence of CME inhibitors, AuNC-BBN uptake in cells is reduced up to 84%. TEM images further confirm CME characteristic clathrin coated pits and lysosomal release of AuNCs. These results demonstrate that peptide ligands conjugated to the surface of nanoparticles maintain their target specificity. This bolsters the case for peptide robustness and its persisting functionality in intracellular vehicular delivery systems.

DIETARY SILVER NANOPARTICLES REDUCE FITNESS IN A BENEFICIAL, BUT NOT PEST, INSECT SPECIES.

Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.

Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to KRAS mutant NSCLC dissociates GAB1-SHP2 post oncogene knockdown

A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation.

Three-Dimensional Nanocomposites: Fluidics Driven Assembly of Metal Nanoparticles on Protein Nanostructures and Their Cell-Line-Dependent Intracellular Trafficking Pattern

Three-dimensional nanocomposites prepared using two different families of nanomaterials holds significant relevance pertaining to biological applications. However, integration of the two distinct nanomaterials with precision to control the overall compositional homogeneity of the resulting 3D nanocomposite is a synthetic challenge. Conventional reactions result in nanocomposites with heterogeneous composition and render useless. To address this challenge, we have developed a fluidics-mediated process for controlling the interaction of nanoparticles to yield a compositional uniform multidimensional nanoparticle; as an example, we demonstrated the integration of gold nanoparticles on gelatin nanoparticles. The composition of the nanocomposite is controlled by reacting predetermined number of gold nanoparticles to a known number of thiolated gelatin nanoparticles at any given time within a defined cross-sectional area. Using the fluidics process, we developed nanocomposites of different composition: [gelatin nanoparticles-(gold nanoparticles)x] where xaverage = 2, 12, or 25. The nanocomposites were further surface conjugated with organic molecules such as fluorescent dye or polyethylene glycol (PEG) molecules. To study the biological behavior of nanocomposite, we investigated the cellular internalization and trafficking characteristics of nanocomposites in two human cancer cell lines. The nanocomposites exhibited a three-stage cellular release mechanism that enables the translocation of gold nanoparticles within various cellular compartments. In summary, the three-dimensional nanocomposite serves as a novel platform for developing well-defined protein-metal nanocomposites for potential drug delivery, sensory, and molecular imaging applications.

Anti-corrosive films on silver plasmonic gratings for fluorescence imaging of single molecules and cancer cells

Silver plasmonic gratings with a thin corrosion protection film enable enhanced fluorescence-based detection, including single molecule, over a much wider fluorescent dye concentration range, 100 μM-1fM, and deeper field penetration than traditional sensor substrates.

Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to Kras mutant NSCLC reveals Gab1 assisted survival pathway post oncogene knockdown

are highly magnetic, and stable in serum solutions for extended periods of time. The MNPs are incubated with cells in 1X PBS for 3h for receptor binding at 37 C after which particles bound to cells are magnetically separated with a pull force of 57 lbs. Cells are then washed and counted using an automated algorithm. Thewhole process has been optimized to a minimum cell population of 100 and can be theoretically reduced to a 2h process which makes this extremely effective for clinical evaluations and straightforward. Similar protocol was used when the cells were spiked in blood plasma and captured. Our data suggests strong correlation between number of A549 cell captured when Herceptin conjugated MNPs are used (96%difference vs HCC827), while Cetuximab conjugated MNPs pull both HCC827 aswell as A549 (31%difference). We expect to reduce the cell capture limit to less than 10 cells in further experiments as required for patient testing. In conclusion, our results show that MNP based sensing allows both cell marker characterization as well as capture simultaneously. The nanocubes allow better characterization of HER2 and EGFR positive metastatic cell subpopulations and provide easier prediction of tumor heterogeneity without resorting to invasive procedures.

Abstract 1903: Personalized diagnostics: Receptor specific gold nanorods used for accurate diagnosis of EGFR expression in human tumor tissues

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: The purpose of this study was to apply the enhanced surface chemistry and imaging properties of gold nanorods to the area of tumor protein diagnostics in order to give an accurate, quantifiable interpretation of a patients biomarker status. The epidermal growth factor receptor (EGFR; HER1) is a cell surface receptor that regulates cell migration, adhesion, and proliferation. EGFR is commonly overexpressed in a variety of cancers. EGFR expression testing has been mandated by insurance companies in many areas before therapy can be given. Current methods of EGFR evaluation have been shown to be unreliable, thus EGFR was chosen as a target of interest for our studies. Methods: Gold nanorods (GNR) of aspect ratio 3:4 were prepared using an established seed-mediated growth method. The GNR were then PEGylated and conjugated with a unique modified peptide that has shown specific affinity for the EGF receptor. The product was characterized to show stability and presence of surface-bound peptide. The compound known as GNR-1070 was used to examine correlations between EGFR expression and gold present on the membranes of human cell lines and paraffin-embedded tissue samples. Results: In cell lines that are known to express EGFR, gold particles were identified bound to the membranes. The amount of bound gold correlated with the degree of EGFR expression of each cell line. Histochemical analysis of paraffin-embedded human tissue lines also correlated with the amount of gold bound to the membranes of the tissues. Conclusion: Gold nanorods conjugated with receptor-specific peptides can be used to give an accurate evaluation of protein expression in human cell lines and tissue samples. Citation Format: Chuck Caldwell, Ajit Zambre, Dhananjay Suresh, Gerald Arthur, Raghuraman Kannan. Personalized diagnostics: Receptor specific gold nanorods used for accurate diagnosis of EGFR expression in human tumor tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1903. doi:10.1158/1538-7445.AM2014-1903