Metastatic breast cancer is the second leading cause of cancer-related deaths amongst 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. Since previous studies coupled β3 integrin to epithelial-mesenchymal transition (EMT) and metastasis, we exploited β3 integrin as a therapeutic target to treat TNBC by delivering β3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siβ3). Treatment of TNBC cells with ECO/siβ3 was sufficient to effectively silence β3 integrin expression, attenuate TGF-β-mediated EMT and invasion, restore TGF-β-mediated cytostasis, and inhibit 3-dimensional organoid growth. Modification of ECO/siβ3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siβ3 nanoparticles in vivo alleviated primary tumor burden, and more importantly, significantly inhibited metastasis. Mice bearing orthotopic, TGF-β-pre-stimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siβ3 nanoparticles were free of metastases and relapse after primary tumor resection and 4 weeks after release from the treatment, in comparison to untreated mice. Collectively, these results highlight ECO/siβ3 nanoparticles as a promising therapeutic regimen to combat TNBC.

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.

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 72 hours. 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 72 hours 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

Down-regulation of oncogenes associated with multidrug resistance with RNAi has the potential to enhance the efficacy of cancer chemotherapy. Here, we have designed and developed targeted dual pH-sensitive lipid siRNA self-assembly nanoparticles RGD-PEG(HZ)-ECO/siRNA to enhance cytosolic siRNA delivery via systemic administration, to regulate oncogene expression, and to improve the efficacy of cancer chemotherapy. The dual pH-sensitive function of RGD-PEG(HZ)-ECO/siRNA nanoparticles facilitates effective cytosolic siRNA delivery in cancer cells both in vitro and in vivo after systemic injection. Intravenous injection of RGD-PEG(HZ)-ECO/siRNA nanoparticles (1.0 mg-siRNA/kg) results in effective gene silencing for at least a week in MDA-MB-231 tumor. Intravenous injections of RGD-PEG(HZ)-ECO/siRNA specific to eukaryotic translation initiation factor 4E (eIF4E) every 6 days for 6 weeks down-regulate the overexpression eIF4E protein in tumor and resensitize a drug-resistant MDA-MB-231 breast cancer model to paclitaxel, resulting in significant tumor regression at a low dose, with negligible side effects. RGD-PEG(HZ)-ECO/siRNA nanoparticles also result in minimal immunogenicity after repeated injections in immunocompetent mice. Silencing eIF4E with the targeted dual pH-sensitive multifunctional lipid sieIF4E nanoparticles has the potential to re-sensitize drug-resistant breast cancer to chemotherapy.

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.