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.

Folate receptor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and migration.

BACKGROUND: Human antigen R (HuR) is an RNA binding protein that is overexpressed in many human cancers, including lung cancer, and has been shown to regulate the expression of several oncoproteins. Further, HuR overexpression in cancer cells has been associated with poor-prognosis and therapy resistance. Therefore, we hypothesized that targeted inhibition of HuR in cancer cells should suppress several HuR-regulated oncoproteins resulting in an effective anticancer efficacy. To test our hypothesis, in the present study we investigated the efficacy of folate receptor-α (FRA)-targeted DOTAP:Cholesterol lipid nanoparticles carrying HuR siRNA (HuR-FNP) against human lung cancer cells. RESULTS: The therapeutic efficacy of HuR-FNP was tested in FRA overexpressing human H1299 lung cancer cell line and compared to normal lung fibroblast (CCD16) cells that had low to no FRA expression. Physico-chemical characterization studies showed HuR-FNP particle size was 303.3 nm in diameter and had a positive surface charge (+4.3 mV). Gel retardation and serum stability assays showed that the FNPs were efficiently protected siRNA from rapid degradation. FNP uptake was significantly higher in H1299 cells compared to CCD16 cells indicating a receptor-dose effect. The results of competitive inhibition studies in H1299 cells demonstrated that HuR-FNPs were efficiently internalized via FRA-mediated endocytosis. Biologic studies demonstrated HuR-FNP but not C-FNP (control siRNA) induced G1 phase cell-cycle arrest and apoptosis in H1299 cells resulting in significant growth inhibition. Further, HuR-FNP exhibited significantly higher cytotoxicity against H1299 cells than it did against CCD16 cells. The reduction in H1299 cell viability was correlated with a marked decrease in HuR mRNA and protein expression. Further, reduced expression of HuR-regulated oncoproteins (cyclin D1, cyclin E, and Bcl-2) and increased p27 tumor suppressor protein were observed in HuR-FNP-treated H1299 cells but not in C-FNP-treated cells. Finally, cell migration was significantly inhibited in HuR-FNP-treated H1299 cells compared to C-FNP. CONCLUSIONS: Our results demonstrate that HuR is a molecular target for lung cancer therapy and its suppression using HuR-FNP produced significant therapeutic efficacy in vitro.

Tumor-Targeted Dendrimer Nanoparticles for Combinatorial Delivery of siRNA and Chemotherapy for Cancer Treatment.

In current cancer therapy, the combined targeted delivery of treatments is an important method to enhance the therapeutic efficiency and reduce adverse side effects. Dendrimer-based nanoparticles have received considerable attention for multifunctional therapeutic delivery. In this chapter, we describe the methods for encapsulating the chemotherapeutic drug, cisplatin (CDDP), and human antigen R (HuR)-targeted siRNA into dendrimer nanoparticles for folate receptor-targeted delivery. We discuss the methodologies for physical and biological characterization of synthesized multifunctional (Den-PEI-CDDP-HuR-FA) nanoparticles in detail. Physical characterization includes size and charge determination, drug encapsulation and release kinetics, ligand conjugation, etc., and functional characterization involves testing of the nanoparticles for receptor-specific uptake and cytotoxicity on human lung cancer and normal cells. The protocol provided is geared to provide the readers an overview of developing multifunctional dendrimer-based nanoparticles. However, based on the individual's objective and the type of combinatorial drugs to deliver, the protocol may need modifications in achieving maximal efficacy.

Progress in extracellular vesicle biology and their application in cancer medicine.

Under the broader category of extracellular vesicles (EVs), exosomes are now well recognized for their contribution and potential for biomedical research. During the last ten years, numerous technologies for purification and characterization of EVs have been developed. This enhanced knowledge has resulted in the development of novel applications of EVs. This review is an attempt to capture the exponential growth observed in EV science in the last decade and discuss the future potential to improve our understanding of EVs, develop technologies to overcome current limitations, and advance their utility for human benefit, especially in cancer medicine. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Exosomes as drug delivery vehicle and contributor of resistance to anticancer drugs.

Exosomes are small membranous vesicles implicated in intercellular signalling. Through their uncanny ability to carry and deliver donor cellular cargo (biomolecules) to target cells, they exert a profound effect on the regular functioning of healthy cells and play a significant role in pathogenesis and progression of several diseases, including cancer. The composition and number of endogenously circulating exosomes frequently vary, which is often reflective of the pathophysiological status of the cell. Applicability of exosomes derived from normal cells as a drug carrier with or without modifying their intraluminal and surface components are generally tested. Conversely, exosomes also are reported to contribute to resistance towards several anti-cancer therapies. Therefore, it is necessary to carefully evaluate the role of exosomes in cancer progression, resistance and the potential use of exosomes as a delivery vehicle of cancer therapeutics. In this review, we summarize the recent advancements in the exploitation of exosomes as a drug delivery vehicle. We also discuss the role of exosomes in conferring resistance to anti-cancer therapeutics. While this review is focused on cancer, the exosome-based drug delivery and resistance is also applicable to other human diseases.

Combinatorial Nanoparticle Delivery of siRNA and Antineoplastics for Lung Cancer Treatment.

Recent developments in nanotechnology, especially in drug delivery systems, are advanced by featuring novel multifunctional nanoparticles that promise safe, specific, and efficient therapeutic delivery for cancer treatment. Multifunctional nanoparticle-based drug delivery systems enable simultaneous delivery of multiple therapeutic agents for effective combination therapy for cancer. In this chapter, we provide detailed protocols for development and application of a multifunctional nanoparticle system for combinatorial delivery of a chemotherapeutic (cisplatin) and small interfering RNA (siRNA) for human antigen R (HuR) mRNA in cancer cells using a polyamidoamine (PAMAM) dendrimer platform. Protocols for nanoparticle functionalization with folic acid (FA) for targeted delivery of therapeutics toward folate receptor (FR)-overexpressing cancer cells are also described. Further, methods employed for physiochemical and functional characterization of the multifunctional nanoparticle system are discussed in detail. Using the methods described in this chapter, researchers would be able to develop PAMAM dendrimer-based multifunctional nanoparticles for targeted delivery of chemotherapeutics and siRNA combinations. We also provide an example showing the dendrimer-polyethyleneimine-cis-diamminedichloroplatinum-siRNA-folic acid (Den-PEI-CDDP-siRNA-FA) nanoparticle system developed was therapeutically effective toward non-small cell lung cancer (NSCLC) cell lines (H1299 and A549) while exhibiting reduced toxicity to normal lung fibroblast (MRC9) cells.

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.

Exosomes as Theranostics for Lung Cancer.

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.

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 αvß3 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 αvß3 and cautioned against corresponding uptake of toxic drugs, if any at all. Whereas, normal human bronchial epithelial (NHBE) cells with poor integrin αvß3 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.

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.

HuR silencing elicits oxidative stress and DNA damage and sensitizes human triple-negative breast cancer cells to radiotherapy.

HuR is an mRNA-binding protein whose overexpression in cancer cells has been associated with poor prognosis and resistance to therapy. While reports on HuR overexpression contributing to chemoresistance exist, limited information is available on HuR and radioresistance especially in triple-negative breast cancer (TNBC).In this study we investigated the role of HuR in radiation resistance in three TNBC (MDA-MB-231, MDA-MB-468 and Hs578t) cell lines. Endogenous HuR expression was higher in TNBC cells compared to normal cells. siRNA mediated knockdown of HuR (siHuR) markedly reduced HuR mRNA and protein levels compared to scrambled siRNA (siScr) treatment. Further, siHuR treatment sensitized TNBC cells to ionizing radiation at 2 Gy compared to siScr treatment as evidenced by the significant reduction in clonogenic cell survival from 59%, 49%, and 65% in siScr-treated cells to 40%, 33%, and 46% in siHuR-treated MDA-MB-231, MDA-MB-468 and Hs578t cells, respectively. Molecular studies showed increased ROS production and inhibition of thioredoxin reductase (TrxR) in HuR knockdown cells contributed to radiosensitization. Associated with increased ROS production was evidence of increased DNA damage, demonstrated by a significant increase (p < 0.05) in γ-H2AX foci that persisted for up to 24 h in siHuR plus radiation treated cells compared to control cells. Further, comet assay revealed that HuR-silenced cells had larger and longer-lasting tails than control cells, indicating higher levels of DNA damage. In conclusion, our studies demonstrate that HuR knockdown in TNBC cells elicits oxidative stress and DNA damage resulting in radiosensitization.

Nanosomes carrying doxorubicin exhibit potent anticancer activity against human lung cancer cells.

Successful chemotherapeutic intervention for management of lung cancer requires an efficient drug delivery system. Gold nanoparticles (GNPs) can incorporate various therapeutics; however, GNPs have limitations as drug carriers. Nano-sized cellular vesicles like exosomes (Exo) can ferry GNP-therapeutic complexes without causing any particle aggregation or immune response. In the present study, we describe the development and testing of a novel Exo-GNP-based therapeutic delivery system -'nanosomes'- for lung cancer therapy. This system consists of GNPs conjugated to anticancer drug doxorubicin (Dox) by a pH-cleavable bond that is physically loaded onto the exosomes (Exo-GNP-Dox). The therapeutic efficacy of Dox in nanosomes was assessed in H1299 and A549 non-small cell lung cancer cells, normal MRC9 lung fibroblasts, and Dox-sensitive human coronary artery smooth muscle cells (HCASM). The enhanced rate of drug release under acidic conditions, successful uptake of the nanosomes by the recipient cells and the cell viability assays demonstrated that nanosomes exhibit preferential cytotoxicity towards cancer cells and have minimal activity on non-cancerous cells. Finally, the underlying mechanism of cytotoxicity involved ROS-mediated DNA damage. Results from this study mark the establishment of an amenable drug delivery vehicle and highlight the advantages of a natural drug carrier that demonstrates reduced cellular toxicity and efficient delivery of therapeutics to cancer cells.

Tumor-targeted and pH-controlled delivery of doxorubicin using gold nanorods for lung cancer therapy.

BACKGROUND: In lung cancer, the efficacy of conventional chemotherapy is limited due to poor drug accumulation in tumors and nonspecific cytotoxicity. Resolving these issues will increase therapeutic efficacy. METHODS: GNR-Dox-Tf-NPs (gold nanorod-doxorubicin-transferrin-nanoparticles) were prepared by different chemical approaches. The efficacy of these nanoparticles was carried out by cell viability in lung cancer and primary coronary artery smooth muscle cells. The receptor-mediated endocytosis studies were done with human transferrin and desferrioxamine preincubation. The GNR-Dox-Tf nanoparticles induced apoptosis, and DNA damage studies were done by Western blot, H2AX foci, and comet assay. RESULTS: We developed and tested a gold nanorod-based multifunctional nanoparticle system (GNR-Dox-Tf-NP) that carries Dox conjugated to a pH-sensitive linker and is targeted to the transferrin receptor overexpressed in human lung cancer (A549, HCC827) cells. GNR-Dox-Tf-NP underwent physicochemical characterization, specificity assays, tumor uptake studies, and hyperspectral imaging. Biological studies demonstrated that transferrin receptor-mediated uptake of the GNR-Dox-Tf-NP by A549 and HCC827 cells produced increased DNA damage, apoptosis, and cell killing compared with nontargeted GNR-Dox-NP. GNR-Dox-Tf-NP-mediated cytotoxicity was greater (48% A549, 46% HCC827) than GNR-Dox-NP-mediated cytotoxicity (36% A549, 39% HCC827). Further, GNR-Dox-Tf-NP markedly reduced cytotoxicity in normal human coronary artery smooth muscle cells compared with free Dox. CONCLUSION: Thus, GNR-Dox-Tf nanoparticles can selectively target and deliver Dox to lung tumor cells and alleviate free Dox-mediated toxicity to normal cells.