The impact of microfluidic mixing of triblock micelleplexes on in vitro / in vivo gene silencing and intracellular trafficking.

The triblock copolymer polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG) has been shown to spontaneously assemble into nano-sized particulate carriers capable of complexing with nucleic acids for gene delivery. The objective of this study was to investigate micelleplex characteristics, their in vitro and in vivo fate following microfluidic preparation of siRNA nanoparticles compared to the routinely used batch reactor mixing technique. Herein, PEI-PCL-PEG nanoparticles were prepared with batch reactor or microfluidic mixing techniques and characterized by various biochemical assays and in cell culture. Microfluidic nanoparticles showed a reduction of overall particle size as well as a more uniform size distribution when compared to batch reactor pipette mixing. Confocal microscopy, flow cytometry and qRT-PCR displayed the subcellular delivery of the microfluidic formulation and confirmed the ability to achieve mRNA knockdown. Intratracheal instillation of microfluidic formulation resulted in a significantly more efficient (p < 0.05) knockdown of GAPDH compared to treatment with the batch reactor formulation. The use of microfluidic mixing techniques yields an overall smaller and more uniform PEG-PCL-PEI nanoparticle that is able to more efficiently deliver siRNA in vivo. This preparation method may prove to be useful when a scaled up production of well-defined polyplexes is required.

Post-Transcriptional Regulation of the GASC1 Oncogene with Active Tumor-Targeted siRNA-Nanoparticles.

Basal-like breast cancer (BLBC) accounts for the most aggressive types of breast cancer, marked by high rates of relapse and poor prognoses and with no effective clinical therapy yet. Therefore, investigation of new targets and treatment strategies is more than necessary. Here, we identified a receptor that can be targeted in BLBC for efficient and specific siRNA mediated gene knockdown of therapeutically relevant genes such as the histone demethylase GASC1, which is involved in multiple signaling pathways leading to tumorigenesis. Breast cancer and healthy breast cell lines were compared regarding transferrin receptor (TfR) expression via flow cytometry and transferrin binding assays. Nanobioconjugates made of low molecular weight polyethylenimine (LMW-PEI) and transferrin (Tf) were synthesized to contain a bioreducible disulfide bond. siRNA complexation was characterized by condensation assays and dynamic light scattering. Cytotoxicity, transfection efficiency, and the targeting specificity of the conjugates were investigated in TfR positive and negative healthy breast and breast cancer cell lines by flow cytometry, confocal microscopy, RT-PCR, and Western blot. Breast cancer cell lines revealed a significantly higher TfR expression than healthy breast cells. The conjugates efficiently condensed siRNA into particles with 45 nm size at low polymer concentrations, showed no apparent toxicity on different breast cancer cell lines, and had significantly greater transfection and gene knockdown activity on mRNA and protein levels than PEI/siRNA leading to targeted and therapeutic growth inhibition post GASC1 knockdown. The synthesized nanobioconjugates improved the efficiency of gene transfer and targeting specificity in transferrin receptor positive cells but not in cells with basal receptor expression. Therefore, these materials in combination with our newly identified siRNA sequences are promising candidates for therapeutic targeting of hard-to-treat BLBC and are currently further investigated regarding in vivo targeting efficacy and biocompatibility.

Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine.

The use of small interference RNA (siRNA) to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR) is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide) conjugated to cationic polymer branched polyethylenimine (bPEI), optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299) and low TfR expression (A549 and H460). The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP) at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

Pulmonary Delivery of siRNA via Polymeric Vectors as Therapies of Asthma.

Asthma is a chronic inflammatory disease. Despite the fact that current therapies, such as the combination of inhaled corticosteroids and ß2-agonists, can control the symptoms of asthma in most patients, there is still an urgent need for an alternative anti-inflammatory therapy for patients who suffer from severe asthma but lack acceptable response to conventional therapies. Many molecular factors are involved in the inflammatory process in asthma, and thus blocking the function of these factors could efficiently alleviate airway inflammation. RNA interference (RNAi) is often thought to be the answer in the search for more efficient and biocompatible treatments. However, difficulties of efficient delivery of small interference RNA (siRNA), the key factor in RNAi, to target cells and tissues have limited its clinical application. In this review, we summarize cytokines and chemokines, transcription factors, tyrosine kinases, and costimulatory factors that have been reported as targets of siRNA-mediated treatment in experimental asthma. Additionally, we conclude several targeted delivery systems of siRNA to specific cells such as T cells, macrophages, and dendritic cells, which could potentially be applied in asthma therapy.

Influence of oligospermines architecture on their suitability for siRNA delivery.

Spermines are naturally abundant polyamines that partially condense nucleic acids and exhibit the proton-sponge effect in an acidic environment. However, spermines show a limited efficiency for transfecting nucleic acids because of their low molecular weight. Therefore, spermines need to be modified to be used as nonviral vectors for nucleic acids. Here, we synthesized linear bisspermine as well as a linear and dendritic tetraspermine with different molecular architectures. These oligospermines were self-assembled into polyplexes with siRNA. The structure-activity relationship of the oligospermines was evaluated in terms of their efficiency for delivering siRNA into a nonsmall cell lung carcinoma cell line. Oligospermines displayed minimal cytotoxicity but efficient siRNA condensation and showed better stability against polyanions than polyethylenimine. The morphology of the polyplexes was strongly affected by the oligospermine architecture. Linear tetraspermine/siRNA polyplexes showed the best gene-silencing efficiency among the oligospermines tested at both the mRNA and protein expression levels, indicating the most favorable structure for siRNA delivery.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

Ipilimumab, Pembrolizumab, or Nivolumab in Combination with BBI608 in Patients with Advanced Cancers Treated at MD Anderson Cancer Center.

Background: BBI608 is an investigational reactive oxygen species generator that affects several molecular pathways. We investigated BBI608 combined with immune checkpoint inhibitors in patients with advanced cancers. Methods: BBI608 (orally twice daily) was combined with ipilimumab (3 mg/kg IV every 3 weeks); pembrolizumab (2 mg/kg IV every 3 weeks); or nivolumab (3 mg/kg IV every 4 weeks). We assessed the safety, antitumor activity and the pharmacokinetic profile of BBI combined with immunotherapy. Results: From 1/2017 to 3/2017, 12 patients were treated (median age, 54 years; range, 31-78; 6 men). Treatment was overall well tolerated. No dose-limiting toxicity was observed. The most common adverse events were diarrhea (5 patients: grade (G)1-2, n = 3; G3, n = 2) and nausea (4 patients, all G1). Prolonged disease stabilization was noted in five patients treated with BBI608/nivolumab lasting for 12.1, 10.1, 8.0, 7.7 and 7.4 months. The median progression-free survival was 2.73 months. The median overall survival was 7.56 months. Four patients had prolonged overall survival (53.0, 48.7, 51.9 and 48.2 months). Conclusions: Checkpoint inhibitors combined with BBI608 were well tolerated. Several patients had prolonged disease stabilization and overall survival. Prospective studies to elucidate the mechanisms of response and resistance to BBI608 are warranted.

A Method for Targeted Nonviral siRNA Delivery in Cancer and Inflammatory Diseases.

Small interfering RNA (siRNA)-based therapy has been subject of intense research since the discovery of RNA interference (RNAi), providing a tool to potentially silence any chosen gene. Nevertheless, efficient delivery still presents a major hurdle to translating this promising technology into medical practice. Here, we describe a straightforward method to prepare and characterize an effective delivery system consisting of low-molecular-weight polyethylenimine (PEI) and transferrin (Tf). Tf-PEI polyplexes are not only able to successfully transport and protect the sensitive nucleic acid payload from degradation but also to selectively deliver the siRNA to transferrin receptor (TfR)-overexpressing cells, playing key roles in the pathology of numerous cancer types as well as inflammatory diseases.

Evaluating the Regulation of Cytokine Levels After siRNA Treatment in Antigen-Specific Target Cell Populations via Intracellular Staining.

RNA interference (RNAi) offers a promising base for therapeutic knockdown of clinically relevant genes. Local delivery routes as well as targeted delivery to specific cell populations have been shown to circumvent several hurdles of successful siRNA delivery in vivo. To evaluate and quantify the treatment effect in a precise way, next to measuring the downregulation on gene and protein levels, it is equally essential to investigate the influence on downstream factors such as generated cytokines. Here, we describe an expressive method to specifically isolate the desired target cells and determine their levels of intracellular cytokines by flow cytometry using the example of murine lungs after pulmonary in vivo transfection with siRNA.Therefore, the lungs of treated mice are harvested and processed into single cell suspensions, in which CD4 positive T cells are marked by antibody-coupled magnetic beads and isolated via magnetic separation. These purified target cells are then fixed and permeabilized, making their intracellular interleukins accessible for staining with fluorescently labeled antibodies. Thus, the cytokine levels and hence the precise influence of the siRNA treatment on intracellular conditions can be measured.

Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells.

RNA interference (RNAi) offers the potential to selectively silence disease-related genes in defined cell subsets. Translation into the clinical routine is, however, still hampered by the lack of efficient carrier systems for therapeutic siRNA, endosomal entrapment presenting a major hurdle. A promising siRNA delivery system has previously been developed on the base of polyethylenimine (PEI) and the targeting ligand transferrin (Tf) to specifically reach activated T cells in the lung. In the present work, the focus is on optimizing Tf-PEI polyplexes for gene knockdown in primary activated T cells by improving their endosomal escape properties. Blending of the conjugate with membrane lytic melittin significantly enhanced endosomal release and thereby cytoplasmic delivery, while maintaining selective T cell targeting abilities and overall cell tolerability. The gathered data furthermore demonstrate that melittin addition also distinctly improves several other essential particle characteristics, such as siRNA encapsulation efficiency and stability in lung lining fluids. In conclusion, this results in a novel upgraded siRNA delivery system that is not only able to specifically deliver its payload to the desired target cells via receptor-mediated endocytosis, but also shows enhanced release from endosomal vesicles in order to initiate RNAi in the cytoplasm.

Characterization of Dopaminergic System in the Striatum of Young Adult Park2-/- Knockout Rats.

Mutations in parkin gene (Park2) are linked to early-onset autosomal recessive Parkinson's disease (PD) and young-onset sporadic PD. Park2 knockout (PKO) rodents; however, do not display neurodegeneration of the nigrostriatal pathway, suggesting age-dependent compensatory changes. Our goal was to examine dopaminergic (DAergic) system in the striatum of 2 month-old PKO rats in order to characterize compensatory mechanisms that may have occurred within the system. The striata form wild type (WT) and PKO Long Evans male rats were assessed for the levels of DAergic markers, for monoamine oxidase (MAO) A and B activities and levels, and for the levels of their respective preferred substrates, serotonin (5-HT) and ß-phenylethylamine (ß-PEA). The PKO rats displayed lower activities of MAOs and higher levels of ß-PEA in the striatum than their WT counterparts. Decreased levels of ß-PEA receptor, trace amine-associated receptor 1 (TAAR-1), and postsynaptic DA D2 (D2L) receptor accompanied these alterations. Drug-naive PKO rats displayed normal locomotor activity; however, they displayed decreased locomotor response to a low dose of psychostimulant methamphetamine, suggesting altered DAergic neurotransmission in the striatum when challenged with an indirect agonist. Altogether, our findings suggest that 2 month-old PKO male rats have altered DAergic and trace aminergic signaling.

In vitro and in vivo delivery of siRNA via VIPER polymer system to lung cells.

The block copolymer VIPER (virus-inspired polymer for endosomal release) has been reported to be a promising novel delivery system of DNA plasmids both in vitro and in vivo. VIPER is comprised of a polycation segment for condensation of nucleic acids as well as a pH-sensitive segment that exposes the membrane lytic peptide melittin in acidic environments to facilitate endosomal escape. The objective of this study was to investigate VIPER/siRNA polyplex characteristics, and compare their in vitro and in vivo performance with commercially available transfection reagents and a control version of VIPER lacking melittin. VIPER/siRNA polyplexes were formulated and characterized at various charge ratios and shown to be efficiently internalized in cultured cells. Target mRNA knockdown was confirmed by both flow cytometry and qRT-PCR and the kinetics of knockdown was monitored by live cell spinning disk microscopy, revealing knockdown starting by 4 h post-delivery. Intratracheal instillation of VIPER particles formulated with sequence specific siRNA to the lung of mice resulted in a significantly more efficient knockdown of GAPDH compared to treatment with VIPER particles formulated with scrambled sequence siRNA. We also demonstrated using pH-sensitive labels that VIPER particles experience less acidic environments compared to control polyplexes. In summary, VIPER/siRNA polyplexes efficiently deliver siRNA in vivo resulting in robust gene silencing (>75% knockdown) within the lung.

Folate receptor targeted three-layered micelles and hydrogels for gene delivery to activated macrophages.

New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor ß in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation.

Targeted delivery of siRNA to activated T cells via transferrin-polyethylenimine (Tf-PEI) as a potential therapy of asthma.

Asthma is a worldwide health problem. Activated T cells (ATCs) in the lung, particularly T helper 2 cells (Th2), are strongly associated with inducing airway inflammatory responses and chemoattraction of inflammatory cells in asthma. Small interfering RNA (siRNA) as a promising anti-sense molecule can specifically silence inflammation related genes in ATCs, however, lack of safe and efficient siRNA delivery systems limits the application of siRNA as a therapeutic molecule in asthma. Here, we designed a novel pulmonary delivery system of siRNA, transferrin-polyethylenimine (Tf-PEI), to selectively deliver siRNA to ATCs in the lung. Tf-PEI polyplexes demonstrated optimal physicochemical properties such as size, distribution, zeta-potential, and siRNA condensation efficiency. Moreover, in vitro studies showed significantly enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary ATCs. Biodistribution of polyplexes in a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. In conclusion, the present work proves the feasibility to target ATCs in asthma via Tf receptor. This strategy could potentially be used to design an efficient siRNA delivery system for asthma therapy.