Polyplex Delivery Research Articles

Poly(ethylene Glycol) (PEG)–OligoRNA Hybridization to mRNA Enables Fine‐Tuned Polyplex PEGylation for Spleen‐Targeted mRNA Delivery

Organ‐selective targeting of mRNA polyplexes has been rarely explored despite the substantial potential of polymer‐based systems in mRNA delivery. In this study, spleen‐selective delivery of polyplexes is achieved by employing mRNA engineering to coat them with poly(ethylene glycol) (PEG). In this approach, mRNA is hybridized with PEGylated complementary RNA oligonucleotides (PEG–OligoRNAs), followed by the addition of linear poly(ethyleneimine). In this method, it is ensured that nearly all added PEG strands bind to the polyplexes, thereby enabling precise control of PEG amounts on the surface. Following systemic injection into mice, non‐PEGylated polyplexes yield robust protein expression in the lung and spleen. Intriguingly, adding a small number of PEG–OligoRNAs drastically reduces protein expression efficiency in the lung while preserving it in the spleen, realizing spleen targeting of mRNA polyplexes. Furthermore, PEGylated polyplexes demonstrate their potential utility in mRNA vaccination. In mechanistic analyses, non‐PEGylated polyplexes immediately agglomerate in the blood and deposit in the lung. Coating polyplexes with a small amount of short PEG effectively prevents these processes. Notably, even slight changes in PEG amounts and lengths dramatically impact the physicochemical properties and biological functionalities of the polyplexes, emphasizing the benefits of an mRNA engineering‐based approach for fine‐tuning polyplex PEG coating.

Investigation of charge ratio variation in mRNA – DEAE-dextran polyplex delivery systems

mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required.In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) – Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application.

Delivery of pDNA Polyplexes to Bronchial and Alveolar Epithelial Cells Using a Mesh Nebulizer.

In this study, a cell penetrating peptide was used as an uptake enhancer for pDNA delivery to the lungs. Polyplexes were prepared between pDNA and CPP. Intracellular delivery of pDNA was assessed in both alveolar (A549) and bronchial (Calu-3) epithelial cells. Aerosol delivery was investigated using a mesh nebulizer. Efficient intracellular delivery of pDNA occurs in both A549 and Calu-3 cells when delivered as polyplexes. Protection against nucleases and endosomal escape mechanism occurs when pDNA is formulated within the polyplexes. For aerosol delivery, 1% (w/v) mannitol was able to protect naked DNA structure during nebulization with a significant increase in fine particle fraction (particles <5μm). The structure of polyplexes when delivered via a mesh nebulizer using 1% (w/v) mannitol could partially withstand the shear forces involved in aerosolization. Although some loss in functionality occurred after nebulization, membrane-associated fluorescence was observed in A549 cells. In Calu-3 cells mucus entrapment was a limiting factor for polyplex delivery. The presence of CPP is essential for efficient intracellular delivery of pDNA. The polyplexes can be delivered to lung epithelial cells using mesh nebulizer. The use of different excipients is essential for further optimization of these delivery systems.

Bioinspired pH- and Temperature-Responsive Injectable Adhesive Hydrogels with Polyplexes Promotes Skin Wound Healing.

Despite great potential, the delivery of genetic materials into cells or tissues of interest remains challenging owing to their susceptibility to nuclease degradation, lack of permeability to the cell membrane, and short in vivo half-life, which severely restrict their widespread use in therapeutics. To surmount these shortcomings, we developed a bioinspired in situ-forming pH- and temperature-sensitive injectable hydrogel depot that could control the delivery of DNA-bearing polyplexes for versatile biomedical applications. A series of multiblock copolymer, comprised of water-soluble poly(ethylene glycol) (PEG) and pH- and temperature-responsive poly(sulfamethazine ester urethane) (PSMEU), has been synthesized as in situ-forming injectable hydrogelators. The free-flowing PEG-PSMEU copolymer sols at high pH and room temperature (pH 8.5, 23 °C) were transformed to stable gel at the body condition (pH 7.4, 37 °C). Physical and mechanical properties of hydrogels, including their degradation rate and viscosity, are elegantly controlled by varying the composition of urethane ester units. Subcutaneous administration of free-flowing PEG-PSMEU copolymer sols to the dorsal region of Sprague-Dawley rats instantly formed hydrogel depot. The degradation of the hydrogel depot was slow at the beginning and found to be bioresorbable after two months. Cationic protein or DNA-bearing polyplex-loaded PEG-PSMEU copolymer sols formed stable gel and controlled its release over 10 days in vivo. Owing to the presence of urethane linkages, the PEG-PSMEU possesses excellent adhesion strength to wide range of surfaces including glass, plastic, and fresh organs. More importantly, the hydrogels effectively adhered on human skin and peeled easily without eliciting an inflammatory response. Subcutaneous implantation of PEG-PSMEU copolymer sols effectively sealed the ruptured skin, which accelerated the wound healing process as observed by the skin appendage morphogenesis. The bioinspired in situ-forming pH- and temperature-sensitive injectable adhesive hydrogel may provide a promising platform for myriad biomedical applications as controlled delivery vehicle, adhesive, and tissue regeneration.

Pulmonary delivery of polyplexes for combined PAI-1 gene silencing and CXCR4 inhibition to treat lung fibrosis

This report describes the development of polyplexes based on CXCR4-inhibiting poly(ethylenimine) derivative (PEI-C) for pulmonary delivery of siRNA to silence plasminogen activator inhibitor-1 (siPAI-1) as a new combination treatment of pulmonary fibrosis (PF). Safety and delivery efficacy of the PEI-C/siPAI-1 polyplexes was investigated in vitro in primary lung fibroblasts isolated from mice with bleomycin-induced PF. Biodistribution analysis following intratracheal administration of fluorescently labeled polyplexes showed prolonged retention in the lungs. Treatment of mice with bleomycin-induced PF using the PEI-C/siPAI-1 polyplexes resulted in a significant down-regulation of the PAI-1 expression and decreased collagen deposition in the lung. The results of this study provide first evidence of the potential benefits of combined inhibition of CXCR4 and PAI-1 in the pulmonary treatment of PF.

One Size Does Not Fit All: The Effect of Chain Length and Charge Density of Poly(ethylene imine) Based Copolymers on Delivery of pDNA, mRNA, and RepRNA Polyplexes.

Nucleic acid delivery systems are commonly translated between different modalities, such as DNA and RNA of varying length and structure, despite physical differences in these molecules that yield disparate delivery efficiency with the same system. Here, we synthesized a library of poly(2-ethyl-2-oxazoline)/poly(ethylene imine) copolymers with varying molar mass and charge densities in order to probe how pDNA, mRNA, and RepRNA polyplex characteristics affect transfection efficiency. The library was utilized in a full factorial design of experiment (DoE) screening, with outputs of luciferase expression, particle size, surface charge, and particle concentration. The optimal copolymer molar mass and charge density was found as 83 kDa/100%, 72 kDa/100%, and 45 kDa/80% for pDNA, RepRNA, and mRNA, respectively. While 10 of the synthesized copolymers enhanced the transfection efficiency of pDNA and mRNA, only 2 copolymers enhanced RepRNA transfection efficiency, indicating a narrow and more stringent design space for RepRNA. These findings suggest that there is not a "one size fits all" polymer for different nucleic acid species.

Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA

Despite the extracellular barriers for siRNA delivery have been overcome by utilizing advanced nanoparticle delivery systems, the key intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape still remains challenging. To address the issues, we developed a unique pH- and redox potential-responsive polyplex delivery system based on the copolymer of mPEG-b-PLA-PHis-ssPEI1.8 k, which is composed of a pH-responsive copolymer of PEG-b-PLA-PHis (Mw 5 k) and a branched PEI (Mw1.8 k) linked with redox cleavable disulfide bond. The copolymer showed excellent siRNA complexation and protection abilities against endogenous substances at the relatively low N/P ratio of 6. The siRNA release from the polyplexes (N/P 6) was markedly increased from 13.62% to 58.67% under conditions simulating the endosomal microenvironment. Fluorescence resonance energy transfer (FRET) test also indicated a higher disassembly extent of siRNA from the copolymer. The accelerated siRNA release from the polyplexes was markedly restrained when the N/P ratio was raised above 10 due to the increasing of electrostatic interactions. The efficient endosomal escape of siRNA after internalization was confirmed by confocal microscopy, which was attributed to the cleavaged PEI chains inducing membrane destabilization, the “proton sponge effect” of PHis and PEI as well as the relative small size of after disassembly. The enhanced disassembly and endosomal escape were elucidated as the leading cause for polyplexes (N/P 6) showed more efficient Bcl-2 silencing (85.45%) than those polyplexes with higher N/P ratios (N/P 10 and 15). In vivo results further demonstrated that polyplexes (N/P 6) delivery of siBcl-2 significantly inhibited the MCF-7 breast tumor growth as compared to its counterparts. The incorporation of convertible non-electrical interactions at a balance with electrostatic interactions in complexation siRNA has been demonstrated as an effective strategy to achieve efficient disassembly from stable polyplexes. Moreover, polyplexes equipped with the enhanced disassembly and endosomal escape provides a new potential way to tackle the intracellular delivery bottleneck for siRNA delivery.

Fluorination Enhances Serum Stability of Bioreducible Poly(amido amine) Polyplexes and Enables Efficient Intravenous siRNA Delivery.

The use of small interfering RNA (siRNA) in cancer treatment has been limited by the lack of effective systemic delivery methods. Although synthetic polycations have been widely explored in siRNA delivery, polycation/siRNA polyplexes often suffer from insufficient stability in vivo. Here, rationally designed siRNA delivery systems that meet the requirements for systemic siRNA delivery to distant tumors are reported. The hypothesis that modular design of delivery systems based on poly(amido amine)s that combine fluorination for systemic stability with bioreducibility for easy intracellular siRNA release, and PEGylation for improved safety and colloidal stability will overcome problems with contradicting siRNA delivery demands is tested. PEGylated, fluorinated, and bioreducible copolymers (PEG-PCD-F) with different degree of fluorination are thus synthesized. The fluorinated copolymers readily formed polyplexes with siRNA and achieved greatly improved gene silencing efficacy in multiple cell lines in vitro when compared with nonfluorinated controls. The results show fluorination-induced enhancement of stability, cellular uptake, and endosomal escape of the polyplexes, while exhibiting efficient siRNA release in reducing intracellular environment. PEG-PCD-F polyplexes with siRNA against Bcl2 inhibit breast tumor growth following systemic intravenous administration. The results provide strong evidence of successful combination of bioreducibility with fluorination and PEGylation to achieve systemic siRNA polyplex delivery.

Self-replicating RNA vaccine functionality modulated by fine-tuning of polyplex delivery vehicle structure

The major limitations with large and complex self-amplifying RNA vaccines (RepRNA) are RNase-sensitivity and inefficient translation in dendritic cells (DCs). Condensing RepRNA with polyethylenimine (PEI) gave positive in vitro readouts, but was largely inferior to virus-like replicon particles (VRP) or direct electroporation. In the present study, we improved such polyplex formulation and determined that fine-tuning of the polyplex structure is essential for ensuring efficacious translation. Thereby, three parameters dominate: (i) PEI molecular weight (MW); (ii) RepRNA:PEI (weight:weight) ratio; and (iii) inclusion of cell penetrating peptides (CPPs). Seven commercially available linear PEIs (MW 2,500–250,000) were classified as strong, intermediate or low for their aptitude at complexing and protecting RepRNA for delivery into porcine blood DCs. Inclusion of (Arg)9 or TAT(57-57) CPPs further modified the translation readouts, but varied for different gene expressions. Dependent on the formulation, translation of the gene of interest (GOI) inserted into the RepRNA (luciferase, or influenza virus hemagglutinin or nucleoprotein) could decrease, while the RepRNA structural gene (E2) translation increased. This was noted in the porcine SK6 cell line, as well as both porcine and, for the first time, human DCs. Two formulations – [Rep/PEI-4,000 (1:3)] and [Rep/PEI-40,000 (1:2)/(Arg)9] were efficacious in vivo in mice and pigs, where specific CD8+ T and CD4+ T-cell responses against the GOI-encoded antigen were observed for the first time. The results demonstrate that different polyplex formulations differ in their interaction with the RepRNA such that only certain genes can be translated. Thus, delivery of these large self-replicating RNA molecules require definition with respect to translation of different genes, rather than just the GOI as is the norm, for identifying optimal delivery for the desired immune activation in vivo.

Efficient delivery of signal-responsive gene carriers for disease-specific gene expression via bubble liposomes and sonoporation

Sonoporation is a promising method to intracellularly deliver synthetic gene carriers that have lower endocytotic uptake than viral carriers. Here, we applied sonoporation to deliver genes via polyethylene glycol (PEG)-grafted polymeric carriers that specifically respond to hyperactivated protein kinase A (PKA). PEG-grafted polymeric carrier/DNA polyplexes were not efficiently delivered into cells via the endocytotic pathway because of the hydrophilic PEG layer surrounding the polyplexes. However, the delivery of polyplexes into cells was significantly increased by sonoporation. The delivered polyplexes exhibited PKA-responsive transgene expression in PKA-overexpressing cells, but not in cells with low PKA activation. These results show that the sonoporation-mediated delivery of PEG-modified PKA-responsive polyplexes is a promising approach for safely applying gene therapy to abnormal cells with hyperactivated PKA.

Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol.

Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA-polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.

Effective humoral immune response from a H1N1 DNA vaccine delivered to the skin by microneedles coated with PLGA-based cationic nanoparticles

Intradermal DNA vaccination is a promising method of immunization that overcomes some practical drawbacks of conventional intramuscular vaccinations. However, it is difficult to deliver DNA vaccines to target cells in the skin and polyplexes. This study outlines the development of an intradermal pH1N1 DNA vaccine delivery platform using microneedles (MNs) coated with a polyplex containing poly lactic-co-glycolic acid/polyethyleneimine (PLGA/PEI) nanoparticles (NPs). Stainless steel MNs with enhanced hydrophilicity have been manufactured by silanization, which improves coating efficiency. MNs coated with the polyplex encapsulating pDNA vaccine were prepared by optimizing the N/P ratio, with a 6:1 ratio showing the highest transfection efficiency in mammalian cells. Polyplexes were coated on MNs without severe aggregation of the polyplex in the dry form. The coated polyplex rapidly dissolved in porcine skin (within 5min) and induced a greater humoral immune response than that of intramuscular polyplex delivery or naked pH1N1 DNA vaccine delivery by a dry-coated MN. These results indicate that intradermal delivery of pDNA vaccines within a cationic polyplex coated on MNs has potential in skin immunizations.

Combining Fluorination and Bioreducibility for Improved siRNA Polyplex Delivery.

Polycations are promising vectors for the delivery of siRNA therapeutics but they often suffer from toxicity and low in vivo delivery efficacy. This study tests the hypothesis that combining fluorination and bioreducibility of polycations will overcome problems with both the toxicity and delivery efficacy. To test the hypothesis, we synthesized bioreducible (RHB) and nonreducible (NHB) poly(amido amine)s. The RHB were additionally fluorinated using reaction with heptafluorobutyric anhydride to obtain F-RHB. We found that both RHB and F-RHB showed significantly reduced cytotoxicity compared with NHB, which allowed their safe use in a wider range of doses than NHB. All three synthesized polycations formed polyplexes with siRNA. F-RHB achieved the best siRNA silencing efficacy in multiple cell lines in vitro, which was at least in part because of fluorination-induced enhancement of cellular uptake and improved endosomal escape. Lastly, F-RHB showed greatly improved Luc silencing efficacy in tumors in vivo when compared with polyplexes based on RHB, NHB, as well as control poly(ethylenimine) (PEI). This study suggests that combining fluorination with bioreducibility of polycations is a promising strategy to the design of siRNA delivery vectors with improved toxicity and in vivo activity profiles.

The neuropilin-1 receptor mediates enhanced tumor delivery of H2K polyplexes.

Promising plasmid-based treatments have limited value without an effective delivery system. Recently, the linear H2K with a repeating -KHHK- pattern was determined to be an effective plasmid carrier to tumor xenografts in vivo. Although unpacking of the H2K polyplex within the tumor may have a role, the mechanism for the enhanced efficacy remains unclear. After solid-phase synthesis of linear and branched histidine-lysine (HK) peptide carriers of plasmids, the peptides were compared for their ability to lyse endosomes with a red blood cell model and to transfect MDA-MB-435 xenografts in the presence or absence of neuropilin-1 receptor (NRP-1) antibodies. To examine stability, polyplexes were incubated with trypsin or NaCl and then analyzed by electrophoresis. After screening peptides with a model for endosomal lysis at two pHs, the 33-mer H3K peptide lysed red blood cells effectively at the lower pH. Combining H3K and H2K peptides as carriers of plasmids expressing luciferase were more effective than H2K alone. Based on the repeating -KHHK- sequences of H2K, we studied whether the widespread gene expression in the tumor may be mediated by NRP-1. By blocking NRP-1 in tumor-bearing mice, luciferase activity in tumors delivered by HK polyplexes was reduced by 96%, whereas activity in normal tissues was minimally reduced. Combining an endosomolytic peptide, H3K, with H2K polyplexes as a carrier further enhanced transfection in vivo. Moreover, the widespread distribution of H2K polyplexes is mediated by NRP-1, suggesting that transcytosis of these polyplexes through the tumor endothelium may lead to efficient transfection. Copyright © 2016 John Wiley & Sons, Ltd.

Application of vasoactive and matrix-modifying drugs can improve polyplex delivery to tumors upon intravenous administration

Low efficacy of cationic polymer-based formulations (polyplexes) for systemic gene delivery to tumors remains the crucial concern for their clinical translation. Here we show that modulating the physiological state of a tumor using clinically approved pharmaceuticals can improve delivery of intravenously injected polyplexes to murine melanoma tumors with different characteristics. Direct comparison of drugs with different mechanisms of action has shown that application of nitroglycerin or losartan improved extravasation and tumor uptake of polyplex nanoparticles, whereas angiotensin II had almost no effect on polyplex accumulation and microdistribution in the tumor tissue. Application of nitroglycerin and losartan caused from 2- to 6-fold enhanced efficacy of polyplex-mediated gene delivery depending on the tumor model. The results obtained on polyplex behavior in tumor tissues depending on physiological state of the tumor can be relevant to optimize delivery of polyplexes and other nanomedicines with similar physicochemical properties.

Exploring zwitterionic siRNA polyplex coronas as an alternative to traditional PEGylated architectures for improved intravenous pharmacokinetics

Event Abstract Back to Event Exploring zwitterionic siRNA polyplex coronas as an alternative to traditional PEGylated architectures for improved intravenous pharmacokinetics Meredith Jackson1, Thomas A. Werfel1, Zoe E. Johnson1, Todd D. Giorgio1 and Craig L. Duvall1 1 Vanderbilt University, Biomedical Engineering, United States Introduction: Despite the use of PEGylation to improve in vivo stability of traditional polymeric siRNA delivery vehicles, circulation half-lives remain short (~20 min) [1]. Serum opsonins and complement proteins adsorb onto these particles, decreasing stability and increasing recognition and uptake by the mononuclear phagocyte system (MPS)[2]-[4]. To improve intravenous polyplex delivery of RNAi therapeutics, it is imperative to design nanocarriers with improved resistance to the challenges of a complex protein environment. The current work compares zwitterionic corona chemistry to traditional linear and branched PEG-based architectures for predictors of best in vivo function. To our knowledge, this work describes and provides initial characterization of the first endosomolytic zwitterion-based polymeric siRNA delivery system. Materials and Methods: Poly[(ethylene glycol)-b-[(2-dimethylamino)ethyl methacrylate) (DMAEMA)-co-(butyl methacrylate) (BMA)] (PEG-DB), poly[(ethyl ether methacrylate) (POEGMA)-b-p(DMAEMA-co-BMA)] (POEGMA-DB), and poly(methacryloyloxyethylphosphorylcholine (PMPC)-b-p(DMAEMA-co-BMA)] (PMPC-DB), were RAFT polymerized with an optimized 50:50 ratio of DMAEMA and BMA[5]. Polymers were characterized with GPC and 1H-NMR. Polyplexes formulated from these polymers and siRNA were characterized with DLS and TEM. These polyplexes were assessed for uptake and in vitro knockdown of the model gene luciferase in breast cancer cells (MDA-MB-231), as well as for endosomal escape function and cytotoxicity. Finally, isothermal titration calorimetry was used to probe adsorption of bovine serum albumin. Results and Discussion: Overall, zwitterionic PMPC-DB notably outperformed PEG-DB and POEGMA-DB in terms of increased cell uptake (Fig 1C) and reduced protein adsorption (Fig 2). Each polymer formed siRNA polyplexes of similar size (~150 nm) and had neutral zeta potential with low polydispersities. All polyplexes exhibit similar knockdown of the model gene luciferase (Fig 1B) and equally low levels of toxicity. Based on isothermal titration calorimetry (Fig 2), zwitterionic PMPC-DB was least favorable to protein adsorption compared to PEG-DB, POEGMA-DB, and a control formulation with a purely cationic DMAEMA corona (D-DPB), as evidenced by its large change in Gibbs free energy (ΔG). However, PMPC-DB had the greatest increase in enthalpy, suggesting that more energy is required to displace the hydration shell of zwitterionic polyplex coatings than for traditional PEG coatings. While the displacement of cage-like hydration shells resulted in favorable entropy changes for PMPC-DB, this effect was outweighed by the enthalpy required to displace them, resulting in higher ΔG values. Conclusion: In conclusion, novel zwitterionic siRNA polyplex coronas show promise for improved in vivo pharmacokinetics relative to conventional PEG based on increased uptake and reduced adsorption of serum proteins. This is a significant finding as there is a known direct correlation between level of opsonization and rapidity of hepatic uptake of IV-injected nanocarriers [4]. In vivo pharmacokinetic effects of the PMPC corona will be compared to PEG in ongoing studies. This work was supported in part by National Science Foundation Graduate Research Fellowship Grant No. 1445197.; We would like to thank members of the Duvall and Giorgio labs for scientific discussions and support regarding this work.; We would like to thank Dr. Joel Tellinghuisen for his guidance regarding isothermal titration calorimetry methods.; This work was supported in part by the NIH through the grant 1R01EB019409.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) – absolute requirements for vaccine design – were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. From the Clinical EditorThe use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Live imaging of transgene expression in Cloudman S91 melanoma cells after polyplex-mediated gene delivery

Utilizing nanoparticles made of cationic polymers as gene carriers is a promising approach in cancer gene therapy. One of the major requirements for successful gene delivery is DNA translocation into the nuclei of cancer cells. Nuclear envelope breakdown during mitosis has been considered as the most favorable opportunity for DNA translocation to the nucleus. Here, we aimed to study the influence of mitosis on polyplex-mediated gene delivery using time-lapse microscopy as a safe and accurate tool. Studying of reporter gene expression on a single cell level enabled to confirm the significance of cell division for gene delivery to Cloudman S91 melanoma cells, in spite of minor mitosis-independent transfection, and to discover some important details of polyplex delivery process. We have found that cell division can result in only one post-mitotic transfected cell of the two that could indicate non-uniform distribution of a very small number of intact plasmid DNA between daughter cells. According to our data, the shorter the time interval from polyplex addition to cell division, the longer time is required for the start of reporter gene expression after completed cytokinesis that presumably is a result of gradual polyplex dissociation in cell. Most probably, the development of new gene delivery carriers which would combine the strong ability to protect DNA and ability to release it during mitosis can provide an increase in intact DNA molecule number per cell, uniform DNA distribution between two post-mitotic cells, and fast reporter gene expression resulting in superior transfection of proliferating cells.

271. Improved Gene Transfer with a New Class of PEG-Peptide Scavenger Receptor Inhibitors

Scavenger receptors (SR) found on Kupffer and fenestrated endothelial cells of the recticuloendothelial system (RES) in liver are responsible for the capture and degradation of both viral1 and non viral gene delivery systems 2. SR inhibition using polyinosinic acid (Poly-I) has been shown to improve the gene transfer efficiency of AdV, AAV, and measles virus by blocking rapid metabolism in the liver. While Poly-I is reportedly not toxic to mice, co-administration of Poly-I with AdV decreases the lethal threshold for AdV in mice by an unknown mechanism1, making Poly-I inhibition of SR a clinically unacceptable approach to inhibit viral uptake by the RES in the liver. In the present study, we report the discovery of a new class of PEGylated polylysine peptides that are potent and safe SR inhibitors. PEGylated(30kDa)-Cys-Trp-Lys(N) (where N = 10, 15, 20, 25, and 30) were prepared by solid phase peptide synthesis and administered to mice i.v. with 125I-pGL3. Administration of 1 nmol of PEG-peptide with 1 mg of 125I-pGL3 resulted in capture of 60% of the polyplex dose by the liver. However, dose-escalation of the PEG-peptide revealed a dose-dependent decrease in 125I-pGL3 PEG-peptide polyplex capture by the liver, resulting from SR inhibition. A maximum dose of 80 nmols of PEG(30kDa)-Cys-Trp-Lys25 inhibited SR mediated uptake of 125I-pGL3 by the liver to 10%. The potency of PEG-peptide inhibition of SR was also dependent on the length of the Lys repeat, with Lys30kDa=Lys25Lys20Lys15 Lys10. Application of delayed hydrodynamic (HD)-stimulation at 1 hour post-pGL3 polyplex delivery resulted in potent gene expression only when administering a SR inhibitory dose (80 nmol) of PEG-peptide. The reported PEG-peptides are proposed to function by in situ formation of 30 nm albumin nanoparticles in the blood that bind to SRs. This new class of safe and potent SR inhibitors may have broad applications by improving both viral and non-viral delivery systems by blocking SR-mediated uptake in the liver.

Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus.

Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair.