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. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

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). These single macromolecules show outstanding aqueous solubility, ultra-high multivalency and biodegradability, making them ideal as nanomedicines. While well-established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation was observed between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half-lives. Real-time analysis of the biodistribution dynamics showed architecture-driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer-based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

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. We sought to verify which of the restrictive dietary approaches is the most potent and if the molecular pathways responsible for the impact of the diets overlap. We characterized the impact of the diets in the context of several dietary restriction-related parameters, including immune status in the GI tract; microbiota and its metabolites; bile acids (BAs); gut morphology; as well as autophagy-, mitochondria-, and energy restriction-related parameters. The effects of the various diets are very similar, particularly between CR, IF, and FMD. The occurrence of a 50 kDa truncated form of occludin, the composition of the microbiota, and BAs distinguished KD from the other diets. Based on the results, we were able to provide a comprehensive picture of the impact of restrictive diets on the gut, indicating that restrictive protocols aimed at improving gut health may be interchangeable.

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. We applied a two-step computational approach with database search and molecular docking to design GE11 variants with improved binding. Synthesized peptides underwent binding studies on immobilized EGFR using surface plasmon resonance. Conjugates of peptides coupled via heterobifunctional PEG linker to linear polyethylenimine (LPEI) were used for transfection studies on EGFR-overexpressing cells using reporter gene encoding plasmid DNA. Docking studies unraveled similarities between GE11 and the EGFR dimerization arm. By skipping non-overlapping amino acids, a less hydrophobic segment (YTPQNVI) was identified to be directly involved in EGFR binding. By replacing valine by tyrosine, a full-length version with proposed enhanced binding (GE11m3) was developed. While hydrophobic or hydrophilic segments and variations thereof exhibited low binding, GE11m3 exhibited 3-fold increase in binding compared to GE11, validating in silico predictions. In transfection studies, polyplexes with GE11m3 induced a significantly higher reporter gene expression when compared to GE11 polyplexes both on murine and human cancer cells overexpressing EGFR.

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. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

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). G2CNN AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE-/- mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2CNN AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2CNN AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2CNN AAV9-gRNAeNOS . Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.

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.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

Surface Modification of E. coli 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 E. coli the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of E. coli 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. By transferring the process to bacterial vesicles, we can generate a hybrid of perfectly eukaryotic proteins (in terms of folding and post-translational modifications) on a prokaryotic platform. We could demonstrate that two different GPI proteins can be displayed on the same OMV. In addition to fluorescent marker proteins, cytokines, growth factors and antigens canb be potentially transferred, generating a versatile modular platform for a novel vaccine strategy.

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 in vitro, delivered dosages in vivo 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). Semitelechelic, thiolated linear polyethylenimine (PEI) was chemisorbed onto gold nanoparticles to endow them with positive charge. A simple two-step complexation method offers tunable loading of siRNA at concentrations relevant for in vivo studies and the flexibility for inclusion of multiple functionalities without any purification steps. SiRNA was electrostatically complexed with these cationic gold nanoparticles and further condensed with polycation or polyethyleneglycol-polycation conjugates. The resulting auropolyplexes ensured complete complexation of siRNA into nanoparticles with a high load of ∼15,500 siRNA molecules/nanoparticle. After efficient internalization into the tumor cell, an 80% knockdown of the luciferase reporter gene was achieved. Auropolyplexes were applied intratracheally in Balb/c mice for pulmonary delivery, and their biodistribution were studied spatio-temporally and quantitatively by optical tomography. Auropolyplexes were well tolerated with ∼25% of the siRNA dose remaining in the lungs after 24 h. Importantly, siRNA was released from auropolyplexes in vivo and a fraction also crossed the air-blood barrier, which was then excreted via kidneys, whereas >97% of gold nanoparticles were retained in the lung. Linear PEI-based auropolyplexes offer a combination of successful endosomal escape and better biocompatibility profile in vivo. Taken together, combined chemisorption and complexation endow auropolyplexes with crucial biophysical attributes, enabling a versatile and upscalable nanogold-based platform for siRNA delivery in vitro and in vivo.

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. A high level of sensitivity was obtained using the luciferase reporter genes for firefly (FLuc), secreted Gaussia (GLuc) and synthetic NanoLuc (NLuc). As fluorescent reporter proteins, mTurqouise2, tdTomato and iRFP720 were chosen. Specificity of pathway activity was validated by co-transfection with pathway activating genes, showing significant response to induction. In addition, multi-gene plasmids were cloned, allowing the detection of all three pathways by one vector. By using the multi-gene vector 3P-Luc (wnt-NLuc, hedgehog-FLuc, Notch-GLuc), we could unambiguously demonstrate the crosstalk between pathways, while excluding cross reactivity of luciferase substrates. First studies with synthetic compounds confirmed the applicability of the system for future drug screening approaches.

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. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.

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 (L5) 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 L5 content (10-20%, w/w), and (ii) the L5:siRNA ratio (10,1-30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3ß-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. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3ß (GSK3ß) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3ß axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3ß.

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. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and pDNA.

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. The method describes preparation of firefly luciferase assay buffer (F-LAB) along with BCA assay and employment of the optimized firefly luciferase assay for investigating in vitro gene delivery by polyplex and lipoplex nanoparticles. Reusability of F-LAB for ease of usage (by freezing and reusing it for luciferase assay) is also demonstrated.

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. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

In vivo 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 in vivo due to photoluminescence. METHODS: Fat grafts in cluster form were labeled with cadmium-telluride (CdTe)-QD 770 and transplanted subcutaneously in a murine in vivo model. Photoluminescence levels were serially followed in vivo. RESULTS: Tracing of fat grafts was possible for 50 days with CdTe-QD 770. The remaining photoluminescence was 4.9%±2.5% for the QDs marked fat grafts after 30 days and 4.2%± 1.7% after 50 days. There was no significant correlation in the relative course of the tracking signal, when vital fat transplants were compared to non-vital graft controls. CONCLUSIONS: For the first-time fat grafts were tracked in vivo with CdTe-QDs. CdTe-QDs could offer a new option for in vivo tracking of fat grafts for at least 50 days, but do not document vitality of the grafts.

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. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

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. Using ATC xenograft mouse models, transfection efficiency by 123I scintigraphy and potential for systemic radioiodine therapy after systemic polyplex application were evaluated. RESULTS: In vitro iodide uptake studies in SW1736 and Hth74 ATC cells, and, for comparison, in more differentiated follicular (FTC-133) and papillary (BCPAP) thyroid carcinoma cells demonstrated high transfection efficiency and EGFR-specificity of LPEI-PEG-GE11/NIS that correlated well with EGFR expression levels. After systemic polyplex injection, in vivo 123I gamma camera imaging revealed significant tumor-specific accumulation of radioiodine in an SW1736 and an Hth74 xenograft mouse model. Radioiodine accumulation was found to be higher in SW1736 tumors, reflecting in vitro results, EGFR expression levels, and results from ex vivo analysis of NIS staining. Administration of 131I in LPEI-PEG-GE11/NIS-treated SW1736 xenograft mice resulted in significantly reduced tumor growth associated with prolonged survival compared to control animals. CONCLUSIONS: The data open the exciting prospect of NIS-mediated radionuclide imaging and therapy of ATC after non-viral reintroduction of the NIS gene. The high tumor specificity after systemic application makes the strategy an attractive alternative for the treatment of highly metastatic ATC.

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.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.