Narsireddy Amreddy

Polymeric Nanoparticle-Mediated Gene Delivery for Lung Cancer Treatment

In recent years, researchers have focused on targeted gene therapy for lung cancer, using nanoparticle carriers to overcome the limitations of conventional treatment methods. The main goal of targeted gene therapy is to develop more efficient therapeutic strategies by improving the bioavailability, stability, and target specificity of gene therapeutics and to reduce off-target effects. Polymer-based nanoparticles, an alternative to lipid and inorganic nanoparticles, efficiently carry nucleic acid therapeutics and are stable in vivo. Receptor-targeted delivery is a promising approach that can limit non-specific gene delivery and can be achieved by modifying the polymer nanoparticle surface with specific receptor ligands or antibodies. This review highlights the recent developments in gene delivery using synthetic and natural polymer-based nucleic acid carriers for lung cancer treatment. Various nanoparticle systems based on polymers and polymer combinations are discussed. Further, examples of targeting ligands or moieties used in targeted, polymer-based gene delivery to lung cancer are reviewed.

Chemodrug delivery using integrin-targeted PLGA-Chitosan nanoparticle for lung cancer therapy

In this study, we report the efficacy of RGD (arginine-glycine-aspartic acid) peptide-modified polylactic acid-co-glycolic acid (PLGA)-Chitosan nanoparticle (CSNP) for integrin αvβ3 receptor targeted paclitaxel (PTX) delivery in lung cancer cells and its impact on normal cells. RGD peptide-modified chitosan was synthesized and then coated onto PTX-PLGA nanoparticles prepared by emulsion-solvent evaporation. PTX-PLGA-CSNP-RGD displayed favorable physicochemical properties for a targeted drug delivery system. The PTX-PLGA-CSNP-RGD system showed increased uptake via integrin receptor mediated endocytosis, triggered enhanced apoptosis, and induced G2/M cell cycle arrest and more overall cytotoxicity than its non-targeted counterpart in cancer cells. PTX-PLGA-CSNP-RGD showed less toxicity in lung fibroblasts than in cancer cells, may be attributed to low drug sensitivity, nevertheless the study invited close attention to their transient overexpression of integrin αvβ3 and cautioned against corresponding uptake of toxic drugs, if any at all. Whereas, normal human bronchial epithelial (NHBE) cells with poor integrin αvβ3 expression showed negligible toxicity to PTX-PLGA-CSNP-RGD, at equivalent drug concentrations used in cancer cells. Further, the nanoparticle demonstrated its capacity in targeted delivery of Cisplatin (CDDP), a drug having physicochemical properties different to PTX. Taken together, our study demonstrates that PLGA-CSNP-RGD is a promising nanoplatform for integrin targeted chemotherapeutic delivery to lung cancer.

Tumor-targeted nanoparticle delivery of HuR siRNA inhibits lung tumor growth in vitro and in vivo by disrupting the oncogenic activity of the RNA-binding protein HuR

Selective downregulation of the human antigen R (HuR) protein by siRNA may provide a powerful approach for treating lung cancer. To this end, we investigated the efficacy of transferrin receptor-targeted liposomal nanoparticle-based HuR siRNA (HuR-TfNP) therapy and compared with control siRNA (C)-TfNP therapy both, in vitro and in vivo using lung cancer models. In vitro studies showed HuR-TfNP, but not C-TfNP, efficiently downregulated HuR and HuR-regulated proteins in A549, and HCC827 lung cancer cells, resulting in reduced cell viability, inhibition of cell migration and invasion, and induction of G1 cell-cycle arrest culminating in apoptosis. However, HuR-TfNP activity in normal MRC-9 lung fibroblasts was negligible. In vivo biodistribution study demonstrated that fluorescently labeled HuR-siRNA or ICG dye–loaded TfNP localized in tumor tissues. Efficacy studies showed intratumoral or intravenous administration of HuR-TfNP significantly inhibited A549 (>55% inhibition) and HCC827 (>45% inhibition) subcutaneous tumor growth compared with C-TfNP. Furthermore, HuR-TfNP treatment reduced HuR, Ki67, and CD31 expression and increased caspase-9 and PARP cleavage and TUNEL-positive staining indicative of apoptotic cell death in tumor tissues compared with C-TfNP treatment. The antitumor activity of HuR-TfNP was also observed in an A549-luc lung metastatic model, as significantly fewer tumor nodules (9.5 ± 3.1; P < 0.001; 88% inhibition) were observed in HuR-TfNP–treated group compared with the C-TfNP–treated group (77.7 ± 20.1). Significant reduction in HuR, Ki67, and CD31 expression was also observed in the tumor tissues of HuR-TfNP-treatment compared with C-TfNP treatment. Our findings highlight HuR-TfNP as a promising nanotherapeutic system for lung cancer treatment. Mol Cancer Ther; 16(8); 1470–86. ©2017 AACR.

Recent Advances in Nanoparticle-Based Cancer Drug and Gene Delivery

Effective and safe delivery of anticancer agents is among the major challenges in cancer therapy. The majority of anticancer agents are toxic to normal cells, have poor bioavailability, and lack in vivo stability. Recent advancements in nanotechnology provide safe and efficient drug delivery systems for successful delivery of anticancer agents via nanoparticles. The physicochemical and functional properties of the nanoparticle vary for each of these anticancer agents, including chemotherapeutics, nucleic acid-based therapeutics, small molecule inhibitors, and photodynamic agents. The characteristics of the anticancer agents influence the design and development of nanoparticle carriers. This review focuses on strategies of nanoparticle-based drug delivery for various anticancer agents. Recent advancements in the field are also highlighted, with suitable examples from our own research efforts and from the literature.

Chemo-biologic combinatorial drug delivery using folate receptor-targeted dendrimer nanoparticles for lung cancer treatment

Co-administration of functionally distinct anti-cancer agents has emerged as an efficient strategy in lung cancer treatment. However, a specially designed drug delivery system is required to co-encapsulate functionally different agents, such as a combination of siRNA and chemotherapy, for targeted delivery. We developed a folic acid (FA)-conjugated polyamidoamine dendrimer (Den)-based nanoparticle (NP) system for co-delivery of siRNA against HuR mRNA (HuR siRNA) and cis-diamine platinum (CDDP) to folate receptor-α (FRA) -overexpressing H1299 lung cancer cells. The co-delivery of HuR siRNA and CDDP using the FRA-targeted NP had a significantly greater therapeutic effect than did individual therapeutics. Further, the FRA-targeted NP exhibited improved cytotoxicity compared to non-targeted NP against lung cancer cells. Finally, the NP showed negligible toxicity towards normal MRC9 lung fibroblast cells. Thus, the present study demonstrates FRA-targeted Den nanoparticle system as a suitable carrier for targeted co-delivery of siRNA and chemotherapy agents in lung cancer cells.

Nanoparticles for siRNA-Based Gene Silencing in Tumor Therapy

Gene silencing through RNA interference (RNAi) has emerged as a potential strategy in manipulating cancer causing genes by complementary base-pairing mechanism. Small interfering RNA (siRNA) is an important RNAi tool that has found significant application in cancer therapy. However due to lack of stability, poor cellular uptake and high probability of loss-of-function due to degradation, siRNA therapeutic strategies seek safe and efficient delivery vehicles for in vivo applications. The current review discusses various nanoparticle systems currently used for siRNA delivery for cancer therapy, with emphasis on liposome based gene delivery systems. The discussion also includes various methods availed to improve nanoparticle based-siRNA delivery with target specificity and superior efficiency. Further this review describes challenges and perspectives on the development of safe and efficient nanoparticle based-siRNA-delivery systems for cancer therapy.

Exosomes as Theranostics for Lung Cancer

Abstract Extensive research in genetics and genomics has revealed that lung cancer is a physiologically complex and genetically heterogeneous disease. Although molecular targets that can yield favorable response have been identified, those targets cannot be exploited due to the lack of suitable drug carriers. Furthermore, lung cancer often is diagnosed at an advanced stage when the disease has metastasized. Conventional treatments are not effective for treating metastatic lung cancer. Targeted therapeutics while beneficial has challenges that include poor tumor‐targeting, off‐target effects, and development of resistance to therapy. Therefore, improved drug delivery systems that can deliver drugs specifically to tumor will produce improved treatment outcomes. Exosomes have a natural ability to carry functional biomolecules, such as small RNAs, DNAs, and proteins, in their lumen. This property makes exosomes attractive for use in drug delivery and molecular diagnosis. Moreover, exosomes can be attached to nanoparticles and used for high precision imaging. Exosomes are now considered an important component in liquid biopsy assessments, which are useful for detecting cancers, including lung cancer. Several studies are currently underway to develop methods of exploiting exosomes for use as efficient drug delivery vehicles and to develop novel diagnostic modalities. This chapter summarizes the current status of exosome studies with regard to their use as theranostics in lung cancer. Examples from other cancers have also been cited to illustrate the extensive applicability of exosomes to therapy and diagnosis.

Exosome RNAs as Biomarkers and Targets for Cancer Therapy

Abstract Exosomes are now realized as important contributors in cellular communication, which can transport many biological molecules and perturb physiological changes in the recipient cells. Several researchers have suggested that the content of exosomes changes when they are released from cells under physiological (i.e., disease) or physical stress. Because noncoding RNAs, such as miRNA (miRNA) and long noncoding RNA, form a major component of exosome cargo, they have been regarded as a vital source of biomarkers and therapeutic cargo. This chapter discusses in detail the noncoding RNAs present in the exosome and the literature exploring the use of exosome miRNAs as biomarkers for diagnosis and treatment response in major cancer types. The technology and newer methods of studying exosome RNAs are also reviewed.

489. Tumor-Targeted Hursirna-Nanoparticle Delivery Inhibits Lung Tumor Growth In Vitro and In Vivo

HuR, an mRNA binding protein regulates the stability of many oncoproteins associated with cell survival, proliferation, migration and angiogenesis. HuR overexpression is a marker for poor prognosis in patients diagnosed with cancer of lung, ovary, breast and colon. We hypothesized that the silencing of HuR using small interfering RNA (siRNA) could be a promising approach for lung cancer therapy. To test our hypothesis, we developed a tumor-targeted nanoparticle (NP) system that is targeted to transferrin receptor (TfR) for delivering HuRsiRNA (HuR-TfNP) in human lung cancer cells. Human lung cancer cells (A549, HCC827) and normal lung fibroblast (MRC-9) cell lines expressing varying levels of TfR were used in the present study. TfR expression was highest in A549, moderate in HCC827, and low to undetectable in MRC9 cells. In vitro studies demonstrated enhanced uptake of Tf-NP (51%) in TfR overexpressing A549 cells, compared to the non-targeted NP. Specificity studies using desferrioxamine (DFO; 100 µM), a stimulator of TfR, showed a two-fold increased uptake of Tf-NP whereas blocking TfR with exogenous transferrin (1 µg/well) reduced the uptake by 3 fold in A549 cells. Further, HuR-TfNP treatment reduced HuR expression and significantly suppressed cell proliferation at 24h and 48h compared to control siRNA containing NP (C-Tf-NP) in tumor cells but not in normal cells. Greatest inhibition was observed in A549 cells (23% and 30% inhibition at 24 and 48 h respectively) compared to 15% and 25% in HCC827. In MRC-9 cells, only 4% inhibition was observed. HuR-TfNP induced G1 cell-cycle arrest in tumor cells that correlated with marked reduction in Cyclin D1, and Cyclin E protein expression. Further, tumor cell migration and invasion was significantly inhibited in HuR-TfNP treated tumor cells compared to C-TfNP treatment (p<0.001). In-vivo, Tf-NP bio-distribution studies using indocyanine green (ICG) showed accumulation of the NP in tumor tissues over time with maximum accumulation at 24 h post NP injection. Efficacy studies in A549 tumor model demonstrated that systemic administration of HuR-TfNP significantly inhibited growth of both subcutaneous tumor growth and experimental lung metastasis compared to C-TfNP treatment (P<0.05). Further, tumor growth delay was sustained over 70 days when compared to control groups. A marked reduction in the expression of HuR and HuR-regulated oncoproteins (Bcl2, Cyclin D1 and Cyclin E) with a concomitant increase in p27 expression was observed in HuR-TfNP-treated tumors compared to control tumors. Our study results demonstrate HuR-TfNP therapy suppressed lung tumor growth both in vitro and in vivo and is therapeutic target for lung cancer treatment.

Abstract 3306: The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress

The RNA-binding protein human antigen R (HuR) associates with U-/AU-rich mRNAs encoding proteins that control cell proliferation, metabolism and the stress response. HuR is overexpressed in several human cancers and its overexpression is associated with poor prognosis and resistance to therapy. While the role of HuR in drug resistance has been studied, its contribution to radiation resistance has not been examined. Therefore, we investigated the role of HuR in radiation resistance of triple negative breast cancer (TNBC) cells: MDA-MB-231, MDA-MB-468 and Hs578t. Reduction of HuR expression using small interfering (si) RNA decreased cell proliferation and sensitized TNBC cells to ionizing radiation. Clonogenic assays indicated that silencing HuR suppressed the clonogenic survival of all three TNBC cell lines with survival at 2 Gy (SF2) reduced from 59%, 49%, 65% in control cells to 40%, 33%, and 46% in siHuR-treated MDA-MB-231, MDA-MB-468 and Hs578t cells, respectively. To delineate the underlying mechanism of radiosensitization and to identify candidate mRNAs showing altered levels after silencing HuR, we undertook a ribonomic approach. First, since ionizing radiation enhances the production of reactive oxygen species (ROS), causing DNA damage, we investigated the possible involvement of ROS in siHuR-mediated radiosensitization. ROS production in control or HuR-silenced cells treated with or without radiation was measured using the fluorescent dye 2′-7′-Dichlorodihydrofluorescein diacetate (DCFDA). Radiation significantly increased ROS generation in HuR knockdown cells compared to control cells. To further test the involvement of ROS in radiosensitivity, control and HuR-silenced cells were pre-treated with N-Acetyl-L- cysteine (NAC), an ROS scavenger, prior to radiation. The presence of NAC completely prevented radiation sensitivity and ROS production, indicating the involvement of ROS in HuR-mediated radiation sensitivity. Second, we directly tested the involvement of the DNA damage response (DDR) pathway in radiosensitivity after silencing HuR by evaluating the number of γ-H2AX foci (a common indicator of DNA damage) in control and HuR-silenced cells following irradiation. Our results showed that the number of γ-H2AX foci was significantly greater in HuR-silenced cells than in control cells at 1 h, 2 h and 24 h after irradiation. The persistence of γ-H2AX foci suggests that radiosensitization by HuR silencing involves inhibition of the repair of damaged DNA. This hypothesis was supported by the comet assay, which showed that HuR-silenced cells had larger and longer-lasting tails than control cells, in keeping with the higher levels of DNA damage seen after silencing HuR. Our studies indicate that radiosensitization upon HuR knockdown is linked to suppression of the cellular response to genotoxic and oxidative damage. Citation Format: Meghna Mehta, James Griffith, Kanthesh Basalingappa, Anish Babu, Narsireddy Amreddy, Ranganayaki Muralidharan, Myriam Gorospe, Terence Herman, Wei-Qun Ding, Rajagopal Ramesh, Anupama Munshi. The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2015-3306