Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process.

Degradable Bottlebrush Polypeptides and the Impact of their Architecture on Cell Uptake, Pharmacokinetics, and Biodistribution In Vivo.

Bottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non-degradable and thus unsuitable for systemic therapeutic administration. Herein, we report the design and synthesis of novel poly(organo)phosphazene-g-poly(α-glutamate) (PPz-g-PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15-50 nm).

A Comparison of the Impact of Restrictive Diets on the Gastrointestinal Tract of Mice.

The rate of gut inflammatory diseases is growing in modern society. Previously, we showed that caloric restriction (CR) shapes gut microbiota composition and diminishes the expression of inflammatory factors along the gastrointestinal (GI) tract. The current project aimed to assess whether prominent dietary restrictive approaches, including intermittent fasting (IF), fasting-mimicking diet (FMD), and ketogenic diet (KD) have a similar effect as CR.

Structure-based peptide ligand design for improved epidermal growth factor receptor targeted gene delivery.

The epidermal growth factor receptor EGFR allows targeted delivery of macromolecular drugs to tumors. Its ligand, epidermal growth factor, binds EGFR with high affinity but acts mitogenic. Non-mitogenic peptides are utilized as targeting ligands, like the dodecapeptide GE11, although its low binding affinity warrants improvement.

Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation.

Endothelial Retargeting of AAV9 In Vivo.

Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2 AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity.

CD47-targeted cancer immunogene therapy: Secreted SIRPα-Fc fusion protein eradicates tumors by macrophage and NK cell activation.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.

Surface Modification of Outer Membrane Vesicles with Glycosylphosphatidylinositol-Anchored Proteins: Generating Pro/Eukaryote Chimera Constructs.

Extracellular vesicles produced by different types of cells have recently attracted great attention, not only for their role in physiology and pathology, but also because of the emerging applications in gene therapy, vaccine production and diagnostics. Less well known than their eukaryotic counterpart, also bacteria produce extracellular vesicles, in the case of the Gram-negative the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of OMVs with glycosylphosphatidylinositol (GPI)-anchored protein, exploiting a process variably described as molecular painting or protein engineering in eukaryotic membranes, whereby the lipid part of the GPI anchor inserts in cell membranes.

Combined Chemisorption and Complexation Generate siRNA Nanocarriers with Biophysics Optimized for Efficient Gene Knockdown and Air-Blood Barrier Crossing.

Current nucleic acid (NA) nanotherapeutic approaches face challenges because of shortcomings such as limited control on loading efficiency, complex formulation procedure involving purification steps, low load of NA cargo per nanoparticle, endosomal trapping, and hampered release inside the cell. When combined, these factors significantly limit the amount of biologically active NA delivered per cell , delivered dosages for a prolonged biological effect, and the upscalability potential, thereby warranting early consideration in the design and developmental phase. Here, we report a versatile nanotherapeutic platform, termed auropolyplexes, for improved and efficient delivery of small interfering RNA (siRNA).

Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway.

Tracking the activity of signalling pathways is a fundamental method for basic science, as well as in cancer- and pharmaceutical research. The developmental pathways Wnt, Hedgehog and Notch are frequently deregulated in cancers and represent a valuable target for the discovery of novel anticancer compounds. Here we present reporter systems for tracking activity of these pathways by using specific promoter elements driving the expression of either sensitive luciferases or fluorescent proteins.

Peptide-Targeted Polyplexes for Aerosol-Mediated Gene Delivery to CD49f-Overexpressing Tumor Lesions in Lung.

Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin α6) is frequently upregulated in metastasizing, highly aggressive tumors.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration.

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L content (10-20%, w/w), and (ii) the L:siRNA ratio (10,1-30:1, w/w).

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3β-MCL-1 Axis.

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia.

Firefly Luciferase-Based Reporter Gene Assay for Investigating Nanoparticle-Mediated Nucleic Acid Delivery.

Investigation of nanoparticle-mediated nucleic acid delivery is a key step for the development of nucleic acid-based nanotherapeutics. Using luciferases as reporter genes, the efficiency of gene delivery can be quantified in a highly sensitive way based on bioluminescence measurements. Here we describe a robust assay to quantify the activity of exogenously produced firefly luciferase and its normalization by the total protein amount (bicinchoninic acid assay, BCA) in the cells.

Synthesis of Polyethylenimine-Based Nanocarriers for Systemic Tumor Targeting of Nucleic Acids.

Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of protein- or peptide-based ligands can form nanosized polyplexes with plasmid DNA (pDNA) or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of LPEI from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine.

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis.

tracking of adipose tissue grafts with cadmium-telluride quantum dots.

Background: Fat grafting, or lipofilling, represent frequent clinically used entities. The fate of these transplants is still not predictable, whereas only few animal models are available for further research. Quantum dots (QDs) are semiconductor nanocrystals which can be conveniently tracked due to photoluminescence.

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2.

Reintroducing the Sodium-Iodide Symporter to Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC), the most aggressive form of thyroid cancer, is unresponsive to radioiodine therapy. The current study aimed to extend the diagnostic and therapeutic application of radioiodine beyond the treatment of differentiated thyroid cancer by targeting the functional sodium-iodide symporter (NIS) to ATC.

Methods: The study employed nanoparticle vectors (polyplexes) based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG) and coupled to the synthetic peptide GE11 as an epidermal growth factor receptor (EGFR)-specific ligand in order to target a NIS-expressing plasmid (LPEI-PEG-GE11/NIS) to EGFR overexpressing human thyroid carcinoma cell lines.

Multimodal Fluorescence and Bioluminescence Imaging Reveals Transfection Potential of Intratracheally Administered Polyplexes for Breast Cancer Lung Metastases.

Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.

Systemic tumor-targeted sodium iodide symporter (NIS) gene therapy of hepatocellular carcinoma mediated by B6 peptide polyplexes.

Background: Nonviral polymer-based gene transfer represents an adaptable system for tumor-targeted gene therapy because various design strategies of shuttle systems, together with the mechanistic concept of active tumor targeting, lead to improved gene delivery vectors resulting in higher tumor specificity, efficacy and safety.

Methods: Using the sodium iodide symporter (NIS) as a theranostic gene, nonviral gene delivery vehicles based on linear polyethylenimine (LPEI), polyethylene glycol (PEG) and coupled to the synthetic peptide B6 (LPEI-PEG-B6), which specifically binds to tumor cells, were investigated in a hepatocellular carcinoma xenograft model for tumor selectivity and transduction efficiency.

Results: In vitro incubation of three different tumor cell lines with LPEI-PEG-B6/NIS resulted in significant increase in iodide uptake activity compared to untargeted and empty vectors.

Imaging and targeted therapy of pancreatic ductal adenocarcinoma using the theranostic sodium iodide symporter (NIS) gene.

The theranostic sodium iodide symporter (NIS) gene allows detailed molecular imaging of transgene expression and application of therapeutic radionuclides. As a crucial step towards clinical application, we investigated tumor specificity and transfection efficiency of epidermal growth factor receptor (EGFR)-targeted polyplexes as systemic NIS gene delivery vehicles in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) that closely reflects human disease. PDAC was induced in mice by pancreas-specific activation of constitutively active KrasG12D and deletion of Trp53.

Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells.

Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L.

Fluorescence- and computed tomography for assessing the biodistribution of siRNA after intratracheal application in mice.

Pulmonary delivery of nucleic acids opens the possibility for direct treatment of lung diseases, like fibrosis, cancer, and infections. Lung retention and biodistribution of nucleic acids remain important issues for the development of suitable therapeutic approaches. Moreover, monitoring the dynamic biodistribution processes of siRNA after aerosol delivery can help in identifying bottlenecks and optimizing therapeutic concepts.

Cadmium Telluride Quantum Dots as a Fluorescence Marker for Adipose Tissue Grafts.

Plastic and reconstructive surgeons increasingly apply adipose tissue grafting in a clinical setting, although the anticipation of graft survival is insecure. There are only few tools for tracking transplanted fat grafts in vivo.Murine adipose tissue clusters were incubated with negatively charged, mercaptoproprionic acid-coated cadmium telluride quantum dots (QDs) emitting in the dark red or near infrared.