Endosomal Entrapment Research Articles

Abstract 3616: Towards intracellular targeting: Cytosolic delivery using TAT-trimers

Abstract Large therapeutic biomolecules, particularly antibodies, are generally directed against cancer cell surface antigens to facilitate antibody-antigen interaction. This limitation excludes a rich array of potential intracellular cancer targets, which has prompted research to solve the problem of transport of large molecules across the cell membrane and their release into the cytoplasm. However, current efforts suffer from endosomal entrapment and lack of efficacy due to low intracellular concentration ranges. Using the archetypal cell penetrating peptide (CPP) TAT, we report the design, synthesis and evaluation of two novel trimeric TAT clusters with enhanced efficiency in the low micromolar range. In addition, we demonstrate that geometry and conformation of TAT clusters affects internalization properties and mechanism. TAT-trimers were synthesized using copper catalyzed 2+3 cycloaddition between tetrakis core structures and azide / alkyne modified TAT (49-57) peptide. The trimeric clusters were furnished with AlexaFluor488 (AF488) for in vitro detection. Tri-TAT A is designed around a smaller, more strained core structure than tri-TAT B, which is designed around a larger core structure and more flexible. Both trimers, as well as monomeric AF488 labelled TAT (mono-TAT), were evaluated for their cell penetrating properties using live-cell fluorescence confocal microscopy in HeLa and CHO cells. The uptake of tri-TAT A and tri-TAT B into HeLa and CHO was found to be significantly higher than that of mono-TAT (1 μM; 60 min); in addition, the uptake of the trimers (1 μM) was found to be significantly higher than that of 10 μM mono-TAT, indicating that clustering TAT peptides improves their efficacy. Interestingly, we found that tri-TAT A shows homogenous cytosolic uptake and nucleolar staining at 1 μM, while tri-TAT B shows no evidence of cytosolic uptake or nucleolar staining in either HeLa or CHO cells. The fluorescence signal in cells treated with tri-TAT B was focal in nature, which is typical of endosomal entrapment. These results suggest that the geometry of the core structure and the conformational flexibility of TAT trimers is of decisive importance to the efficacy of cytosolic uptake. Time course experiments with tri-TAT A indicate that the homogenous cytosolic signal spreads evenly from the membrane into the cytosol, suggesting that the trimer translocates directly across the membrane, rather than or in addition to entry into the cytosol via endosomal escape. TAT-trimers were successfully conjugated to antibodies and antibody fragments (Fab). These constructs showed favourable internalisation properties in preliminary cell uptake studies in comparison single TAT constructs. Trimeric arrangement of CPPs on a rigid scaffold represents a promising approach to promoting cell uptake of anticancer drugs directed against intracellular targets and a significant improvement over recently reported CPP approaches. Citation Format: Ole Tietz, Rod Chalk, Katherine A. Vallis. Towards intracellular targeting: Cytosolic delivery using TAT-trimers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3616.

Membrane fusogenic high-density lipoprotein nanoparticles

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.

Small, Traceable, Endosome-Disrupting, and Bioresponsive Click Nanogels Fabricated via Microfluidics for CD44-Targeted Cytoplasmic Delivery of Therapeutic Proteins.

Nanogels (NG) are among the most ideal cytoplasmic protein delivery vehicles; however, their performance is suboptimal, partly owing to relatively big size, poor cell uptake, and endosomal entrapment. Here, we developed small, traceable, endosome-disrupting, and bioresponsive hyaluronic acid NG (HA-NG) for CD44-targeted intracellular delivery of therapeutic proteins. With microfluidics and catalyst-free photo-click cross-linking, HA-NG with hydrodynamic diameters of ca. 80 and 150 nm, strong green fluorescence and efficient loading of various proteins including saporin (Sap), cytochrome C, herceptin, immunoglobulin G (IgG), and bovine serum albumin could be fabricated. Interestingly, 80 nm-sized HA-NG revealed clearly better cellular uptake than its 150 nm counterparts in both CD44-negative U87 cancer cells and CD44-positive 4T1 and MDA-MB-231 cells. Moreover, small NG exhibited accelerated endosomal escape, which was further boosted by introducing GALA, a pH-sensitive fusogenic peptide. Accordingly, Sap-loaded small and GALA-functionalized HA-NG showed the highest cytotoxicity in CD44-positive MDA-MB-231, 4T1, A549, and SMMC-7721 cancer cells. The biodistribution studies demonstrated that 80 nm-sized HA-NG displayed significantly greater tumor uptake as well as penetration in MDA-MB-231 human breast tumor xenografts than its 150 nm counterparts, whereas the introduction of GALA had no detrimental effect on tumor accumulation. Small, endosome-disrupting, and bioresponsive HA-NG with easy and controlled fabrication hold a great potential for targeted protein therapy.

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.

Increasing Efficiency of Inducing Pluripotency with a Novel Cell Penetrating Peptide‐Adaptor System

Somatic stem cells have been used to treat a number of disorders for decades but are limited in their ability to differentiate and self‐renew. Induced pluripotent stem cells (iPSCs) not only have the ability to generate cells of all three germ layers, but bypass the ethical concerns related to embryonic stem cells. They also hold the prospect of utilizing a patient's own cells, thus avoiding host‐vs‐graft issues. In 2006, Takahashi and Yamanaka discovered that expression of only four genes were required to induce pluripotency: Oct4, Sox2, Klf4 and c‐Myc (OSKM). Since then, pluripotency has been induced most effectively by transfection or transduction of these genes. However, efficiencies are low, expression is difficult to control, and off‐target effects are a concern. Inducing pluripotency by exogenous delivery of OSKM proteins using cell‐penetrating peptides (CPPs), while promising, is also inefficient due to of poor penetration and entrapment in endosomes. Our novel CPP‐adaptor system overcomes entrapment by relying on the calcium flux during endosomal trafficking. The adaptor, TAT‐CaM, is a recombinant protein consisting of commonly used CPP, TAT, fused to calmodulin. The protein of interest (the ‘cargo’) has an engineered calmodulin binding site (CBS). TAT‐CaM binds purified CBS‐cargo outside the cell where the Ca2+ concentration is relatively high, but after CPP‐mediated endocytosis, cargo dissociates from TAT‐CaM inside endosomes as calcium concentrations drop, leaving the CPP‐adaptor trapped but cargo free to escape to the cytoplasm and beyond. We have used our adaptor system to deliver submicromolar doses of CBS‐Oct4 protein, inducing pluripotency as measured by expression of stem state markers and alkaline phosphatase staining. CBS‐Sox2 and CBS‐Klf4 have also been delivered and while insufficient to induce pluripotency, play an efficiency enhancing role. While not directly comparable to published results, CPP‐mediated efficiency as measured expression of stem‐state markers is higher than current transfection‐ and transduction‐mediated methods. Parameters such as concentration and timing of doses and molar ratios of OSK to each other and TAT‐CaM affect efficiency. iPSCs created via our CPP‐adaptor system can be cultured, expanded over multiple passages and differentiated into other cell types such as cardiomyocytes. Success in this work will lead to a virus‐ and nucleic acid‐free, nontoxic, tunable method for inducing pluripotency.Support or Funding InformationThis work was supported by NIH grant R15GM120691 and R15NS100632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Limitations of Collagen/CPP Hybrid Peptides as Carriers for Cancer Drugs to FaDu Cells.

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.

A Smart pH-Sensitive Delivery System for Enhanced Anticancer Efficacy via Paclitaxel Endosomal Escape.

Micelles are highly attractive nano-drug delivery systems for targeted cancer therapy. While they have been demonstrated to significantly alleviate the side-effects of their cargo drugs, the therapy outcomes are usually suboptimal partially due to ineffective drug release and endosome entrapment. Stimulus-responsive nanoparticles have allowed controlled drug release in a smart fashion, and we want to use this concept to design novel micelles. Herein, we reported pH-sensitive paclitaxel (PTX)-loaded poly (ethylene glycol)-phenylhydrazone-dilaurate (PEG-BHyd-dC12) micelles (PEG-BHyd-dC12/PTX). The micelles were spherical, with an average particle size of ∼135 nm and a uniform size distribution. The pH-responsive properties of the micelles were certified by both colloidal stability and drug release profile, where the particle size was strikingly increased accompanied by faster drug release as pH decreased from 7.4 to 5.5. As a result, the micelles exhibited much stronger cytotoxicity than the pH-insensitive counterpart micelles against various types of cancer cells due to the hydrolysis of the building block polymers and subsequent rapid PTX release. Overall, these results demonstrate that the PEG-BHyd-dC12 micelle is a promising drug delivery system for cancer therapy.

Overcoming Endosomal Entrapment in Drug Delivery.

Intracellular delivery of biological agents such as peptides, proteins, and nucleic acids generally rely on the endocytic pathway as the major uptake mechanism, resulting in their entrapment inside the endosome and lysosome. The recent discovery of cell-penetrating molecules of exceptionally high endosomal escape and cytosolic delivery efficiencies and elucidation of their mechanism of action represent major breakthroughs in this field. In this Topical Review, we provide an overview of the recent progress in understanding and enhancing the endosomal escape process and the new opportunities opened up by these recent findings.

Reactive Oxygen Species (ROS)-Responsive Charge-Switchable Nanocarriers for Gene Therapy of Metastatic Cancer.

The application of nonviral gene vectors has been limited by their insufficient transfection efficiency because of poor serum stability, high endosomal entrapment, limited intracellular release, and low accumulation in the targeted organelle. It is still challenging to design gene carriers with properties that can overcome all of the barriers. We previously developed a reactive oxygen species (ROS)-responsive cationic polymer, poly[(2-acryloyl)ethyl( p-boronic acid benzyl) diethylammonium bromide] (B-PDEAEA), which switches the charge at high concentrations of intracellular ROS to promote intracellular DNA release. However, its gene-delivery efficiency has been limited by serum instability and lysosomal trapping, and coating with an anionic PEGylated lipid only showed mild enhancement. Herein, we coated the ROS-responsive B-PDEAEA polymer with two cationic lipids to form ROS-responsive lipopolyplexes with integrated properties to overcome multiple delivery barriers. The surface cationic lipids endowed the nanocarrier with improved serum stability, effective cellular uptake, and lysosomal evasion. The interior B-PDEAEA/DNA polyplexes, which were highly stable in the extracellular environment, but quickly dissociated, released DNA, promoted nuclei localization, and achieved efficient transcription. The mechanisms of the ROS-responsive and charge-switchable properties of B-PDEAEA were quantitatively studied. The transfection efficiency and antitumor activity of lipopolyplexes were studied in vitro and in vivo. We found that the ROS-responsive lipopolyplexes effectively delivered therapeutic genes into cell nuclei and caused high tumor inhibition in mice bearing peritoneal or lung metastases.

Endosomal Escape and Cytosolic Penetration of Macromolecules Mediated by Synthetic Delivery Agents.

Cell delivery reagents often exploit the endocytic pathway as a route of cell entry. Once endocytosed, these reagents must overcome endosomal entrapment to ensure the release of their macromolecular cargo into the cytosol of cells. In this review, we describe several examples of prototypical synthetic reagents that are capable of endosomal escape and examine their mechanisms of action, their efficiencies, and their effects on cells. Although these delivery systems are chemically distinct, some commonalities in how they interact with cellular membranes can be inferred. This, in turn, sheds some light on the process of endosomal escape, and may help guide the development and optimization of next-generation delivery tools.

Cyclic peptide-based nanostructures as efficient siRNA carriers

RNA interference shows a great strategy for biological studies; however, delivering of small interfering RNA (siRNA) remains challenging. Although several delivery vehicles, including cell-penetrating peptides, have been developed, their implementation is often restricted because of their endosomal entrapment. Herein, we report the formation of self-assembled nanostructures from rationally designed cyclic peptides and explore them for efficient delivery of functional biomacromolecules such as siRNA into mammalian cells. The newly obtained soft materials make stable complexes with siRNAs, thereby increasing their stability and deliver fluorescent labelled siRNA inside the cells as evident from confocal microscopy analysis. Flow cytometry analysis reveals that significant uptake of FAM-siRNA occurs in the presence of peptide nanostructures compared with siRNA alone. Peptide nanostructure-mediated delivery of very low concentration of siRNA causes significant knockdown of the target gene as observed at protein level by Western blot analysis, which is comparable to lipofectamine, commercially available transfection agent.

Smart Polymersomes Dually Functionalized with cRGD and Fusogenic GALA Peptides Enable Specific and High-Efficiency Cytosolic Delivery of Apoptotic Proteins.

Low cell selectivity and uptake coupled with endosomal entrapment pose critical hurdles for intracellular delivery and clinical translation of therapeutic proteins. Herein, we report that smart polymersomes dually functionalized with cRGD and fusogenic GALA peptides (cRGD/GALA-Ps) enable ανβ3-specific and high-efficiency cytosolic delivery of cytochrome C (CC), a model apoptotic protein, to A549 human lung cancer cells. cRGD/GALA-Ps was prepared with 20 mol % cRGD and varying GALA contents from 2 to 4 to 6 mol % via coassembly of PEG- b-poly(trimethylene carbonate- co-dithiolane trimethylene carbonate)-spermine (PEG- b-P(TMC- co-DTC)-spermine), cRGD-PEG- b-P(TMC- co-DTC), and maleimide-PEG- b-P(TMC- co-DTC) and postmodification using GALA-SH (sequence: CWEAALAEALAEALAEHLAEALAEALEALAA). cRGD/GALA-Ps loaded with ∼13 wt % CC displayed a small size of about 65 nm and fast glutathione-triggered protein release. Interestingly, cRGD/GALA-Ps maintained a similar targeting ability to cRGD-Ps in ανβ3-positive A549 lung cancer cells, while markedly enhanced cytosolic release of FITC-labeled CC, as revealed by confocal microscopy. MTT assays exhibited that CC-loaded cRGD/GALA-Ps was significantly more potent than CC-loaded cRGD-Ps, in which cell viabilities of 76.2, 51.0, 29.6, and 35.5% were discerned for cRGD/GALA-Ps with 0, 2, 4, and 6 mol % GALA, respectively, at 15.4 μM CC. Apoptosis assays corroborated that cRGD/GALA-Ps-CC with 4 mol % GALA induced better apoptosis of A549 cells than cRGD-Ps-CC (cell apoptosis: 36.4 vs 14.4%). These results highlight that dual-functionalization of polymersomes with targeting and fusogenic peptides provides an appealing strategy for cytosolic protein delivery.

Molecular mechanisms influencing efficiency of RNA interference in insects.

RNA interference (RNAi) is an endogenous, sequence-specific gene-silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry.

Abstract 5610: CD125xCNE bispecific antibody development to treat bladder cancer

Abstract Background: Bladder cancer is one of the most prevalent cancers impacting adults worldwide.Although,successfully treated in its early stages, there are no effective therapies when the cancer has progressed into the surrounding muscle layer, clinically classified as muscle invasive bladder cancer (MIBC). CD125 was recently shown to be involved in MIBC progression and uniquely overexpressed in MIBC tumors relative to healthy urothelium or superficial tumors. Cyclin E (CNE) is a critical cell cycle protein and regulates progression of normal cells to replicate their DNA. There is a strong link between CNE dysregulation and tumorigenesis and was recently found to be overexpressed in MIBC. We characterized the monoclonal antibodies (mAbs) A14 specific for CD125, and HE-12 and HE-172 specific for CNE with a long-term purpose to develop a CD125xCNE therapeutic bispecific antibody. In order, for the antibody to efficiently target CNEour group developed a natural composite compound (termed Accum)that conjugates to surface lysines and enables mAbs to escape endosomal entrapment followed by active routing to and efficient accumulation in the nucleus. Methods: For Accum conjugation, maleimide groups were introduced into A14 by reaction with 10-to-100-fold molar excess of a PEGylated SMCC crosslinker at RT for 1 h. Purified and concentrated maleimide-derivatized A14 was reacted with 100-fold molar excess Accum for 18 h at 4 °C. Excess Accum-A14 was purified and concentrated. Conjugation was characterized by SDS-PAGE. CD125-positive MIBC cells were treated with Accum-A14 and nuclear localization efficiency determined by confocal microscopy.HE-12 and HE-172 mAbs were evaluated for binding nuclear CNE by flow cytometry using the whole cell and fractionated cell lysate along with saponin to enable the mAbs to diffuse into the cell. In addition, HE-12 and HE-172 mAb binding was evaluated by co-immunoprecipitation. PCR amplification of A14, HE-12, and HE-172 was performed. Results: A14 is readily loaded with Accum with minimal aggregation.Accum-A14 undergoesCD125-specific internalization and has 25-60-fold increased nuclear localization relative to A14 in MIBC cells.The binding of the intracellular CNE by mAb HE-12 and HE-172 was specific. Conclusion: This preliminary data demonstrates that Accum modification of A14 retains CD125 specificity and efficiently localizes to the nucleus of MIBC cells. HE-12 and HE172 recognized CNE. On going work will construct the CD125xCNE bispecific antibody. Future studies will determine the efficiency to target CNE and its impact to MIBC progression. Citation Format: Judit Hunyadkurti, Jeffrey Leyton, Laurent Fafard-Couture, Vincent Lacasse, Marc-Andre Bonin, Angel Lopez. CD125xCNE bispecific antibody development to treat bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5610.

Abstract 756: Development of next-generation antibody-drug conjugates for resistant HER2-positive tumors

Abstract Antibody-Drug Conjugates (ADC) represent a promising therapeutic modality for improving the clinical management of cancer. However, there now exists resistant mechanisms by cancer cells that are effective at ultimately reducing the cellular accumulation of the delivered drug and hence, reducing cytotoxic effectiveness. Kadcyla® (trastuzumab-emtansine [T-DM1]) is a benchmark as it is the first solid-tumor approved ADC. Despite this new therapeutic option, approximately 43% of patients achieve complete or partial response and most who initially respond develop resistance soon after. In order to transform current ADCs to more effective products improved intracellular delivery strategies could provide solutions. Our group developed is a natural composite compound (termed Accum) that conjugates to surface lysines and enables antibodies to escape endosomal entrapment followed by active routing to the nucleus. Modified antibodies retain high tumor cell selectivity, enhanced cellular accumulation, and improved tumor delivery of molecular payloads in vivo. We asked whether the modification of Accum-modified T-DM1 maintained its intracellular delivery mechanism and cytotoxic effectiveness in T-DM1-resistant cells. The cancer cell lines N87, OE19 (gastric), JIMT1, SK-BR-3 (breast), SKOV3 and SKOV3.IP1 (ovarian) were treated with increasing concentrations of T-DM1 in a 3 day on, 10 days off fashion for a period of 1 to 2 months. Resistant clones were identified. Parental and resistant cells were placed and passaged in SILAC (Stably Labeled Amino acid for Cell culture) media for proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Cells were also treated with Accum-T-DM1 and mechanistic underpinnings determined. The IC50 of Accum-T-DM1 was increased 50- and 10-fold relative to T-DM1 in parental OE19, and JIMT1 cells, respectively. T-DM1 resistant cell lines were successfully generated for each tumor type. Typically, the T-DM1 IC50 values were increased ≥100-fold in resistant cells compared to parental cells. Preliminary proteomic studies revealed HER2 expression is marginally reduced in resistant cells. IP of Accum-T-DM1 co-precipitated showed the interaction with α-importin. This study is a work in progress of modifying T-DM1 with Accum and demonstrating enhanced cytotoxicity in T-DM1 resistant HER2-positive tumor cells. Ongoing studies will determine DM1 accumulation levels, potential resistance mechanisms, and ultimately cytotoxic effectiveness. Citation Format: Vincent Lacasse, Jeffrey V. Leyton, Simon Beaudoin, Mark Barok, Heikki Joensuu. Development of next-generation antibody-drug conjugates for resistant HER2-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 756.

An inhibitor screen identifies histone-modifying enzymes as mediators of polymer-mediated transgene expression from plasmid DNA

Effective transgene expression in mammalian cells relies on successful delivery, cytoplasmic trafficking, and nuclear translocation of the delivered vector, but delivery is impeded by several formidable physicochemical barriers on the surface of and within the target cell. Although methods to overcome cellular exclusion and endosomal entrapment have been studied extensively, strategies to overcome inefficient nuclear entry and subsequent intranuclear barriers to effective transient gene expression have only been sparsely explored. In particular, the role of nuclear packaging of DNA with histone proteins, which governs endogenous gene expression, has not been extensively elucidated in the case of exogenously delivered plasmids. In this work, a parallel screen of small molecule inhibitors of chromatin-modifying enzymes resulted in the identification of class I/II HDACs, sirtuins, LSD1, HATs, and the methyltransferases EZH2 and MLL as targets whose inhibition led to the enhancement of transgene expression following polymer-mediated delivery of plasmid DNA. Quantitative PCR studies revealed that HDAC inhibition enhances the amount of plasmid DNA delivered to the nucleus in UMUC3 human bladder cancer cells. Native chromatin immunoprecipitation (N-ChIP)-qPCR experiments in CHO-K1 cells indicated that plasmids indeed interact with intracellular core Histone H3, and inhibitors of HDAC and LSD1 proteins are able to modulate this interaction. Pair-wise treatments of effective inhibitors led to synergistic enhancement of transgene expression to varying extents in both cell types. Our results demonstrate that the ability to modulate enzymes that play a role in epigenetic processes can enhance the efficacy of non-viral gene delivery, resulting in significant implications for gene therapy and industrial biotechnology.

Extrahepatic Targeting of Oligonucleotides with Receptor-Binding Non-Immunoglobulin Scaffold Proteins.

Although recent clinical successes of antisense, splice-switching, and siRNA oligonucleotides have established the therapeutic utility of this novel class of medicines, the efficient systemic application for non-liver targets remains elusive. Exploitation of active receptor-mediated targeting followed by efficient and productive cellular uptake is required for enabling the therapy of extrahepatic diseases on the expressional level. Evasion of liver accumulation and organ-specific targeting and also efficient cytosolic delivery after endosomal internalization are currently insufficiently solved issues. Lipid and polymer-based nanoparticles can be engineered for efficient cellular uptake and enhancement of endosomal escape, but are characterized by preferential liver accumulation based on biodistribution largely determined by particle size and biophysical properties. Oligonucleotide bioconjugates with receptor-binding ligands have been evolved for highly efficient targeting, but frequently result in a large extent of endosomal entrapment and consequently a lack of sufficient cytosolic concentrations. Non-immunoglobulin protein-based receptor recognition affords high cell-type selectivity and is promising for achieving nonhepatic oligonucleotide targeting. The use of such novel protein scaffolds, including designed ankyrin repeat proteins (DARPins), for oligonucleotide delivery is attractive for achieving effective tissue targeting. Issues for further development and optimization to advance approaches for extrahepatic oligonucleotide delivery by nanoparticles or bioconjugates are discussed.

New Approach in Characterize siRNA Delivery Platform for Clinical Translation

Study ObjectivesA cationic peptide‐based nanotechnology system for siRNA delivery recently developed in our lab not only overcomes limitations of serum instability, endosomal entrapment and vascular constraint at the disease sites of interest, but also exhibits an exceptional safety profile (Hou et al, ACS Nano and Biomaterials; 2014). This delivery system has been applied in mechanistic and preclinical studies in the fields of cardiovascular diseases (Vendrov AE, J Am Heart Assoc. 2017; Lozhkin A, J Mol Cell Cardiol 2017) and arthritis (Yan, PNAS 2016; Zhou JCI 2014) with ongoing anti‐cancer applications on more than 6 different malignancies. Here we sought to leverage biophysical approaches to delineate certain critical physical properties responsible in part for the success of this siRNA delivery system.Methods and ResultsTotal reflectance Fourier‐transform infrared spectroscopy (ATR‐FTIR) was used for the first time to demonstrate efficient packing of siRNA into peptide‐based nanoparticles. siRNA and p5RHH have different characteristic absorbance bands within the near IR spectrum, 1700 – 1500 cm−1 for p5RHH and 1250 −1000 cm−1 for siRNA. A net increase in these regions' band intensities corresponds to changes in local concentration of p5RHH/RNA on the FTIR‐ATR crystal. At 6°C and 400:4 μM p5RHH:siRNa ratios, nanoparticles of ~ 355 ± 141 nm radius form and then sediment to the bottom of test tubes (DLS measurements, data not shown). The local increased concentration on the FTIR crystal is due to the slow settling of nanoparticles where the measured spectrum represents a true read out of the nanoparticles composition with minimal contribution from the bulk material. Figure 1 demonstrates dominant bands in the spectrum in the 1700 −1500 cm−1 region (Amide I and II), while siRNA shows low (due to low concentration used) intensity (but distinguishable) peaks in the 1250 −1000 cm−1 with an additional small peak around 1710 cm−1, corresponding to the C=O vibration of guanine. Absorbance bands within these regions exhibit 3 (p5RHH) and 8 (siRNa) fold augmented peaks and the ~1710 cm−1 peak emerges upon nanoparticle formation and settling down on the ATR crystal, strongly indicating a high RNA content in the nanoparticles.ConclusionThis approach confirms siRNA‐peptide condensation and provides evidence‐based guidance for optimizing these and other nanoparticle formulations for more quantitative real time monitoring of siRNA‐peptide complex formation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N′-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.

Convenient preparation of charge-adaptive chitosan nanomedicines for extended blood circulation and accelerated endosomal escape

A major impediment in the development of chitosan nanoparticles (CTS NPs) as effective drug delivery vesicles is their rapid clearance from blood and endosome entrapment. To overcome these problems, a convenient and promising template system was developed by decorating poly(methacrylic acid) (PMAA) to the surface of 10-hydroxy camptothecin (HCPT)-loaded CTS NPs (HCPT-CTS/PMAA NPs). The results show that the presence of negatively charged PMAA significantly elongated the blood circulation time of HCPT-CTS NPs from 12 to 24 h, and reduced the blood clearance (Cl) from 30.57 to 6.72 mL/h in vivo. The calculated area under curve (AUC0-24h) and terminal elimination half-life (t1/2) of HCPT-CTS/PMAA NPs were 4.37-fold and 2.48-fold compared with those of HCPT-CTS NPs. Furthermore, the positively charged HCPT-CTS/PMAA NPs triggered by tumor acidic microenvironment (pH 6.5) result in a 453-fold higher cellular uptake than the negatively charged counterparts at pH 7.4. Additionally, HCPT-CTS/PMAA NPs have the ability to escape endosomal entrapment via “proton sponge effect” after incubation with HepG2 cells for 3 h at pH 6.5. Taken together, these findings open up a convenient, low-cost, but effective way to prepare HCPT-CTS/PMAA NPs as a candidate for developing vectors with enhanced long blood circulation and endosomal escape ability in future clinical experiments.