Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy.

RNAi-mediated knockdown of oncogenes associated with drug resistance can potentially enhance the efficacy of chemotherapy. Here, we have designed and developed targeted dual pH-sensitive lipid-siRNA self-assembly nanoparticles, RGD-PEG(HZ)-ECO/siRNA, which can efficiently silence the oncogene, eukaryotic translation initiation factor 4E (eIF4E), and consequently resensitize triple-negative breast tumors to paclitaxel. The dual pH-sensitive function of these nanoparticles facilitates effective cytosolic siRNA delivery in cancer cells, both in vitro and in vivo. Intravenous injection of RGD-PEG(HZ)-ECO/siRNA nanoparticles (1.0 mg-siRNA/kg) results in effective gene silencing for at least one week in MDA-MB-231 tumors. In addition, treatment of athymic nude mice with RGD-PEG(HZ)-ECO/sieIF4E every 6 days for 6 weeks down-regulates the overexpression of eIF4E and resensitizes paclitaxel-resistant MDA-MB-231 tumors to paclitaxel, resulting in significant tumor regression at a low dose, with negligible side effects. Moreover, repeated injections of the RGD-PEG(HZ)-ECO/siRNA nanoparticles in immunocompetent mice result in minimal immunogenicity, demonstrating their safety and low toxicity. These multifunctional lipid/siRNA nanoparticles constitute a versatile platform of delivery of therapeutic siRNA for treating cancer and other human diseases.

Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect of Silencing HIF-1α with Targeted Multifunctional ECO/siRNA Nanoparticles.

Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced MRI (DCE-MRI) with a biodegradable macromolecular MRI contrast agent was used to noninvasively assess the antiangiogenic effect of RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg of siRNA/kg every 3 days for 3 weeks compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silencing of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in tumor angiogenesis, glycolytic metabolism, and pH regulation. By possessing the ability to elicit a multifaceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment.

Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery.

RNA interference (RNAi) represents a powerful modality for human disease therapy that can regulate gene expression signature using small interfering RNA (siRNA). Successful delivery of siRNA into the cytoplasm of target cells is imperative for efficient RNAi and also constitutes the primary stumbling block in the clinical applicability of RNAi. Significant progress has been made in the development of lipid-based siRNA delivery systems, which have practical advantages like simple chemistry and easy formulation of nanoparticles with siRNA. This review discusses the recent development of pH-sensitive amino lipids, with particular focus on multifunctional pH-sensitive amino lipids for siRNA delivery. The key components of these multifunctional lipids include a protonatable amino head group, distal lipid tails, and two cross-linkable thiol groups, which together facilitate the facile formation of stable siRNA-nanoparticles, easy surface modification for target-specific delivery, endosomal escape in response to the pH decrease during subcellular trafficking, and reductive dissociation of the siRNA-nanoparticles for cytoplasmic release of free siRNA. By virtue of these properties, multifunctional pH-sensitive lipids can mediate efficient cytosolic siRNA delivery and gene silencing. Targeted siRNA nanoparticles can be readily formulated with these lipids, without the need for other helper lipids, to promote systemic delivery of therapeutic siRNAs. Such targeted siRNA nanoparticles have been shown to effectively regulate the expression of cancer-related genes, resulting in significant efficacy in the treatment of aggressive tumors, including metastatic triple negative breast cancer. These multifunctional pH-sensitive lipids constitute a promising platform for the systemic and targeted delivery of therapeutic siRNA for the treatment of human diseases. This review summarizes the structure-property relationship of the multifunctional pH-sensitive lipids and their efficacy in in vitro and in vivo siRNA delivery and gene silencing.

Self-Assembly of a Multifunctional Lipid With Core-Shell Dendrimer DNA Nanoparticles Enhanced Efficient Gene Delivery at Low Charge Ratios into RPE Cells.

Development of safe and effective gene delivery systems is essential in treating ocular genetic disorders. A hybrid nonviral system composed of a multifunctional lipid ECO and a G4 nanoglobule was designed for efficient gene delivery into RPE cells at low charge ratios. This system formed stable DNA nanoparticles at low N/P ratios, exhibited low cytotoxicity, and induced higher GFP expression in ARPE-19 cells at N/P = 6. The hybrid nanoparticles mediated significant reporter gene GFP expression ex-vivo in the retina from wild type C57 mice and in vivo in BALB/c mice. These hybrid nanoparticles are promising for in vitro and in vivo gene delivery at low charge ratios.

Silencing ß3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and Metastasis of Triple-Negative Breast Cancer.

Metastatic breast cancer is the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) is a highly aggressive subcategory of breast cancer and currently lacks well-defined molecular targets for effective targeted therapies. Disease relapse, metastasis, and drug resistance render standard chemotherapy ineffective in the treatment of TNBC. Because previous studies coupled ß3 integrin (ITGB3) to epithelial-mesenchymal transition (EMT) and metastasis, we exploited ß3 integrin as a therapeutic target to treat TNBC by delivering ß3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siß3). Treatment of TNBC cells with ECO/siß3 was sufficient to effectively silence ß3 integrin expression, attenuate TGFß-mediated EMT and invasion, restore TGFß-mediated cytostasis, and inhibit three-dimensional organoid growth. Modification of ECO/siß3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siß3 nanoparticles in vivo alleviated primary tumor burden and, more importantly, significantly inhibited metastasis. In the span of 16 weeks of the experiments and observations, including primary tumor resection at week 9 and release from the treatment for 4 weeks, the mice bearing orthotopic, TGFß-prestimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siß3 nanoparticles were free of metastases and relapse, in comparison with untreated mice. Collectively, these results highlight ECO/siß3 nanoparticles as a promising therapeutic regimen to combat TNBC.

The polyamine catabolic enzyme SAT1 modulates tumorigenesis and radiation response in GBM.

Glioblastoma multiforme (GBM) is the most common and severe form of brain cancer. The median survival time of patients is approximately 12 months due to poor responses to surgery and chemoradiation. To understand the mechanisms involved in radioresistance, we conducted a genetic screen using an shRNA library to identify genes in which inhibition would sensitize cells to radiation. The results were cross-referenced with the Oncomine and Rembrandt databases to focus on genes that are highly expressed in GBM tumors and associated with poor patient outcomes. Spermidine/spermine-N1-acetyltransferase 1 (SAT1), an enzyme involved in polyamine catabolism, was identified as a gene that promotes resistance to ionizing radiation (IR), is overexpressed in brain tumors, and correlates with poor outcomes. Knockdown of SAT1 using shRNA and siRNA approaches in multiple cell and neurosphere lines resulted in sensitization of GBM cells to radiation in colony formation assays and tumors, and decreased tumorigenesis in vivo. Radiosensitization occurred specifically in G2-M and S phases, suggesting a role for SAT1 in homologous recombination (HR) that was confirmed in a DR-GFP reporter system. Mechanistically, we found that SAT1 promotes acetylation of histone H3, suggesting a new role of SAT1 in chromatin remodeling and regulation of gene expression. In particular, SAT1 depletion led to a dramatic reduction in BRCA1 expression, explaining decreased HR capacity. Our findings suggest that the biologic significance of elevated SAT1 expression in GBM lies in its contribution to cell radioresistance and that SAT1 may potentially be a therapeutic target to sensitize GBM to cancer therapies.

Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release.

Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems.

Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery.

Synthetic small interfering RNA (siRNA) has become the basis of a new generation of gene-silencing cancer therapeutics. However, successful implementation of this novel therapy relies on the ability to effectively deliver siRNA into target cells and to prevent degradation of siRNA in lysosomes after endocytosis. In this study, our goal was to design and optimize new amphiphilic cationic lipid carriers that exhibit selective pH-sensitive endosomal membrane disruptive capabilities to allow for the efficient release of their siRNA payload into the cytosol. The pH sensitive siRNA carriers consist of three domains (cationic head, hydrophobic tail, amino acid-based linker). A library of eight lipid carriers were synthesized using solid phase chemistry, and then studied to determine the role of (1) the number of protonable amines and overall pKa of the cationic head group, (2) the degree of unsaturation of the hydrophobic tail, and (3) the presence of histidine residues in the amino acid linker for transfection and silencing efficacy. In vitro screening evaluation of the new carriers demonstrated at least 80% knockdown of a GFP reporter in CHO cells after 72h. The carriers ECO and ECLn performed the best in a luciferase knockdown study in HT29 human colon cancer cells, which were found to be more difficult to transfect. They significantly reduced expression of this reporter to 22.7±3.31% and 23.5±5.11% after 72h post-transfection, better than Lipofectamine RNAiMax. Both ECO and ECLn carriers caused minimal cytotoxicity, preserving relative cell viabilities at 87.3±2.72% and 88.9±6.84%, respectively. A series of hemolysis assays at various pHs revealed that increasing the number of amines in the protonable head group, and removing the histidine residue from the linker, both resulted in improved membrane disruptive activity at the endosomal pH of 6.5. Meanwhile, the cellular uptake into HT29 cancer cells was improved, not only by increasing the amines of the head group, but also by increasing the degree of unsaturation in the lipid tails. Due to flexibility of the synthetic procedure, the delivery system could be modified further for different applications. The success of ECO and ECLn for in vitro siRNA delivery potentially makes them promising candidates for future in vivo studies.

Luminescent charge-transfer complexes: tuning emission in binary fluorophore mixtures.

Charge-transfer (CT) complexes composed of a π-electron-poor naphthalene diimide (NDI) derivative combined with a series of π-electron-rich donors were investigated. Solutions of the CT complexes are nonemissive; however, solid-state complexes and aqueous suspensions display emission that is dependent on the energy of the HOMO of the electron donor. Crystallographic analysis of a pyrene-NDI complex reveals columnar packing and a high degree of frontier molecular orbital (FMO) overlap that likely contributes to the observed optical properties. The fluorescent CT particles are utilized as imaging agents; additional luminescent CT complexes may be realized by considering FMO energies and topologies.

Evidence of preferential pi-stacking: a study of intermolecular and intramolecular charge transfer complexes.

Nine combinations of pi-electron donors and acceptors were examined by UV-vis, fluorescence and (1)H-NMR spectroscopy to identify pi-stacked charge transfer complexes in macromolecular and supramolecular constructs. The high association constant of pyrene and naphthalene diimide suggests a preferentially pi-stacking pair rationalized by frontier orbital congruence.