Polycationic Carriers Research Articles

A Trojan Horse Delivery Vehicle Carrying Sirna Nanotherapeutics with Multiple Tumor Microenvironment Responsiveness Elicits Robust Antitumor Immune Responses in Situ via A "self-synergistic" Approach.

The potential of small interfering RNAs (siRNAs) in the treatment of malignant tumors has attracted increasing attention due to their inherent advantages. However, their therapeutic performance strongly depends on the efficiency of their cytoplasmic delivery in vivo by the delivery vehicle with good cellular permeability and histocompatibility. Herein, a polycationic carrier camouflaged with macrophage membrane was constructed biomimetically, which was condensed from endogenous spermine monomers through diselenide bonds. The developed Trojan horse delivery vehicle has desirable compression efficacy for siPDL1 as well as intracytoplasmic release properties derived from its sequential degradation triggered by redox microenvironment in tumor cells. Further, the co-loading of photosensitizer could mediate PDT accompanied by the generation of reactive oxygen species (ROS) upon light irradiation applied, which accelerated the degradation of the carrier as well as the release of cargoes while enhancing the PDL1 blockage-mediated immunotherapy by inducing in situ immunogenic cell death. Moreover, the synchronously delivered siPDL1 attenuated the ROS-induced increase in immunosuppressive PDL1 expression, thereby effectively eliciting a robust antitumor immune response with a "self-synergistic" manner in the xenograft breast cancer mouse model. This article is protected by copyright. All rights reserved.

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

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

Immunogenicity of mRNA encoding RBD SARS-CoV-2 in complex with a polycationic carrier

BACKGROUND: RBD, receptor-binding domain, a key region of the SARS-CoV-2 surface glycoprotein for virus binding to host cell receptors and one of the targets of virus-neutralizing antibodies. That is why RBD is a promising immunogen for the development of vaccines that can provide protection against COVID-19. Vaccine mRNA is one of the new and rapidly developing vaccine platforms, and the delivery system is a very important component of it.
 AIM: The aim of this work was to present the results of a study of the antigenic properties of mRNA encoding the receptor-binding domain of SARS-CoV-2 when administered in combination with a polycationic carrier.
 MATERIALS AND METHODS: Dynamic and electrophoretic light scattering were used to characterize mRNA complexes with a polyglucin-spermidine conjugate. To assess the immunogenicity of mRNA were immunized BALB/c mice. The specific activity of the sera was assessed using enzyme immunoassay.
 RESULTS: As a result, the sizes and surface charge of the RBD-encoding mRNA complexes with the polyglucin-spermidine conjugate were determined. It has been shown that wrapping mRNA in a polyglucin-spermidine conjugate shell leads to an increase in the induction of RBD-specific antibodies in BALB/c mice compared to naked mRNA.
 CONCLUSIONS: An mRNA encoding the receptor-binding domain of SARS-CoV-2 has been obtained. It has been shown that the packaging of mRNA into the polyglucin-spermidine conjugate shell leads to an increase in immunogenic properties.

Zwitterionic Modification of Polyethyleneimine for Efficient In Vitro siRNA Delivery.

Polyethylenimine (PEI) has been widely used in gene delivery. However, its high cytotoxicity and undesired non-specific protein adsorption hinder the overall delivery efficacy and the practical applications of PEI-based gene delivery systems. In this study, we prepared hydrophobically modified PEIs (H-PEIs) via the reaction of octanal with 40% of primary amines in PEI25k and PEI10k, respectively. Two common zwitterionic molecules, 1,3-propanesultone and β-propiolactone, were then used for the modification of the resulting H-PEIs to construct polycationic gene carriers with zwitterionic properties (H-zPEIs). The siRNA delivery efficiency and cytotoxicity of these materials were evaluated in Hela-Luc and A549-Luc cell lines. Compared with their respective parental H-PEIs, different degrees of zwitterionic modification showed different effects in reducing cytotoxicity and delivery efficiency. All zwitterion-modified PEIs showed excellent siRNA binding capacity, reduced nonspecific protein adsorption, and enhanced stability upon nuclease degradation. It is concluded that zwitterionic molecular modification is an effective method to construct efficient vectors by preventing undesired interactions between polycationic carriers and biomacromolecules. It may offer insights into the modification of other cationic carriers of nucleic acid drugs.

Orientation Identification of Peptide Translocation Through an Aerolysin Nanopore

Nanopores have been proven to have a wide application in biosensing at the single-molecule level. Nanopore-based protein sequencing is getting more and more attention. Recently it has been reported that 13 of the 20 natural amino acids with a short polycationic carrier can be distinguished by aerolysin nanopore via the current blockades, demonstrated that aerolysin nanopore is a suitable tool for protein sequencing. However, there are still more challenges that are needed to overcome while nanopore-based protein sequencing such as the uncertain orientation of the peptide through the pore and the non-uniform charge of the polypeptidic chain, etc. To determine the direction of the protein translocating through the aerolysin nanopore, a mutant aerolysin nanopore was used to detect the Aβ25-35 and Aβ35-25 fragments modified by specific charged tags at C-terminal or N-terminal in our experiments. The results showed that the duration time of N-terminal modified peptide Aβ25-35 and Aβ35-25 is nearly 6 times and 4 times respectively of the C-terminal modified polypeptides at +100 mV, indicating the N-terminal modified peptide have a stronger interaction with aerolysin nanopore. The difference in the interaction behavior between the N-terminal and C-terminal and aerolysin nanopore provides a new idea for designing the threading orientation of peptide for peptide sequencing.

3-Hydroxyhexanoate-based polycationic nanoparticle system for delivering reprogramming factors

Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell.Methods: We have prepared (3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR.Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.

Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore.

Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles.

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.

Rationally Designed Polycationic Carriers for Potent Polymeric siRNA-Mediated Gene Silencing.

The delivery of small interfering RNA (siRNA) remains a major hurdle for the clinical translation of RNA interference (RNAi) therapeutics. Because of its low valency and rigid nature, siRNA typically requires high excesses of cationic delivery materials to package it stably and deliver it to the cytoplasm of target cells, resulting in high toxicities and inefficient gene silencing in vivo. To address these challenges, we pair a polymeric form of siRNA, p-shRNA, with optimized biodegradable polycations to form stable complexes that induce far more potent gene silencing than with siRNA complexes. Furthermore, we unveil a set of design rules governing p-shRNA delivery, using degradable polycations containing hydrophobic and stabilizing polyethylene glycol domains that enable both stable condensation and efficient release inside cells. We demonstrate the therapeutic potential of this approach by silencing the oncogene STAT3 in a well-established B16F10 mouse melanoma model to significantly prolong survival. By blending nucleic acid engineering and polymer design, our system provides a potentially translatable platform for RNAi-based therapies.

Improved siRNA delivery efficiency via solvent-induced condensation of micellar nanoparticles

Efficient delivery of short interfering RNA (siRNA) remains one of the primary challenges of RNA interference therapy. Polyethylene glycol (PEG)ylated polycationic carriers have been widely used for the condensation of DNA and RNA molecules into complex-core micelles. The PEG corona of such nanoparticles can significantly improve their colloidal stability in serum, but PEGylation of the carriers also reduces their condensation capacity, hindering the generation of micellar particles with sufficient complex stability. This presents a particularly significant challenge for packaging siRNA into complex micelles, as it has a much smaller size and more rigid chain structure than DNA plasmids. Here, we report a new method to enhance the condensation of siRNA with PEGylated linear polyethylenimine using organic solvent and to prepare smaller siRNA nanoparticles with a more extended PEG corona and consequently higher stability. As a proof of principle, we have demonstrated the improved gene knockdown efficiency resulting from the reduced siRNA micelle size in mice livers following intravenous administration.

Photolabile Self-Immolative DNA-Drug Nanostructures.

It is often desirable to simultaneously target different cellular pathways to improve the overall efficacy of a drug or to circumvent drug resistance in therapeutic treatments. Nucleic acid therapy has been considered attractive for such combination therapies due to its possible synergistic effects with traditional chemotherapy, especially for targets that do not yet have small molecule inhibitors. However, the co-delivery of nucleic acids and chemotherapeutics typically involves the use of inherently cytotoxic/immunogenic, polycationic carrier systems, for which the benefit is often overshadowed by adverse side effects. Herein, we detail the construction and characterization of a DNA-drug nanostructure that consists almost entirely of payload molecules. Upon triggering with light, the nanostructure collapses via an irreversible, self-immolative process and releases free oligonucleotides, drug molecules, and small molecule fragments. We demonstrate that the nanostructures can be used as a dual-delivery agent in vitro without a carrier system and that the released model drug (camptothecin, CPT) exhibits similar levels of cytotoxicity as unmodified drugs toward cancer cells.

A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich Polycations for Effective Gene Therapy.

For effective gene therapy, nonviral gene carriers with low toxicity and high transfection efficiency are of much importance. In this work, we developed a facile strategy to prepare hyperbranched hydroxyl-rich polycations (denoted by TE) by the one-pot method involving ring-opening reactions between two commonly used reagents, ethylenediamine (ED) with two amino groups and 1,3,5-triglycidyl isocyanurate (TGIC) with three epoxy groups. The hyperbranched TEs with different molecular weights were investigated on their DNA condensation ability, protein absorption property, biocompatibility, transfection efficiency, and in vivo cancer therapy and toxicity. TE exhibited low cytotoxicity and protein absorption property due to the plentiful hydroxyl groups. The optimal transfection efficiency of TE was significantly higher than that of the gold standard polycationic gene carrier branched polyethylenimine (PEI, 25 kDa). Furthermore, TE was applied for in vivo tumor inhibition by the delivery of antioncogene p53, which showed good antitumor efficiency with low adverse effects. The present work provides a new concept for the facile preparation of hyperbranched hydroxyl-rich polycationic carriers with good transfection performances.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Microcarrier-Guided Nanopore Dielectrophoresis for Selective Nucleic Acid Detection

Dielectrophoresis (DEP) is the motion of a polarizable particle in a non-uniform electric field due to an unbalanced electrostatic force on the particle's induced dipole. The DEP mechanism has been extensively utilized for manipulation of biological particles, from cancer cells and viruses to biomolecules such as DNAs and proteins, for their concentration, separation, sorting, and transport. However, current DEP approaches to molecular manipulation are not selective, as DEP is not sensitive enough to discriminate among the induced dipoles of different molecules. Here we explore a novel single-molecule DEP mechanism, carrier-guided nanopore dielectrophoresis, for selective nucleic acid sequence detection. Rather than rely on a target's native polarizability, we designed a polycationic carrier to impart a tunable synthetic dipole to the target nucleic acid molecule; carrier sensitivity and selectivity are both programmable. Such synthetic dipoles can be captured in an engineered nanopore, which acts as an ideal foul-free point source to generate an extremely high field gradient (ΔE∼107 V·m-1 per nanometer or ∼1016 V·m-2) for molecular dipole manipulation. Non-target nucleic acids do not bind the carriers and hence are repelled from the pore by electrophoresis. To elucidate the mechanism of the dipole capture by the nanopore, we took an all-atom molecular dynamics (MD) simulation approach to observe the movements of and forces on the dipole. Simulation results predicted significantly increased force attracting the probe into the engineered nanopore as opposed to the wild type, consistent with the increased capture rates observed in experiment. Most strikingly of all, we find that a carrier with only a few positive charges can drive any length of DNA or RNA with equal capture efficiency. Development of nanopore dielectrophoretic detection thus offers ready medical applications of nanopore technology.

Genetic transformation of moss Ceratodon purpureus by means of polycationic carriers of DNA

There is much progress in application of genetic engineering for improving the biological properties of different organisms. Viral and nonviral carriers are used for delivery of genetic material into target cells. Polymeric materials of natural and synthetic origin are the most promising gene delivery agents. These polymers demonstrated high efficiency of DNA delivery into animal cells, although they were not very effective in plant cells. Here, the procedure for genetic transformation of Ceratodon purpureus (Hedw.) Brid. moss protoplasts is described. The method is based on the application of surface-active polymeric carriers of the poly-DMAEM structure and controlled length and charge. This allows obtaining more transient and stable moss transformants per microgram of plasmid DNA when compared with known protocol based on using polyethyleneglycol. It is easier, more convenient, and cheaper than the “gene gun” method. Prospects for further improvement of structure and functional characteristics of new polymeric carriers are considered for delivery of genetic material into plant cells.

A molecular dynamics simulation study on the effect of lipid substitution on polyethylenimine mediated siRNA complexation

Polycations have been explored as non-viral carriers for effective delivery of small interfering RNA (siRNA). Modifying polycations such as polyethylenimine (PEI) with lipid substitution was found to improve the siRNA delivery efficiency of polycationic carriers. However, the role of such lipid modification is not clear and remains to be probed at the atomistic level. In this work, we elucidate the role of lipid modification through a series of all-atom molecular dynamics simulations on siRNA complexation mediated by a native PEI and four analogous obtained by different lipid modifications. The lipid modification does not affect PEI's capability of neutralizing the siRNA charge, neither does it affect the polyion bridging which plays an important role in siRNA complexation. Significant linkages among the lipid modified PEIs via association of lipid side-groups are observed and this results in more stable and compact PEI/siRNA polyplexes. The lipid associations between short lipids form and break frequently while the lipid associations between long lipids are more stable. For PEIs modified with short lipids, increasing the lipid substitution level results in more compact and stable siRNA structure. For PEIs modified with long lipids, increasing the lipid substitution does not change the amount of PEI linkage via lipid association, and has a reverse effect on compacting siRNA structure due to increased steric hindrance brought by the lipid association on individual PEIs. The simulation results generally correlate well with experimental data and suggest a framework of designing and systematic evaluation of polycation-based siRNA carriers using molecular dynamics simulations.

Polymerized spermine as a novel polycationic nucleic acid carrier system

Spermine, an endogenous amino-group bearing monomer that condenses DNA in sperm, was used as the basic building block to form polycationic nucleic acid carriers via condensation with one of three linker molecules – bischloroformate, succinyl chloride, and glyoxal. The three cationic polymers, polyspermine carbamate (PSP-Carb), polyspermine amide (PSP-Amide) and polyspermine imine (PSP-Imine) were examined for their degradability, cytotoxicity, ability to condense nucleic acids to nanoparticles, and ability to transfect genes or siRNA to cells. PSP-Carb and PSP-Amide exhibited a half-life of more than 2 months when incubated in aqueous buffers at 37°C, while the half-life of PSP-Imine was 11h. Relative cytotoxicity of the polymers, as measured by COS-7 and HepG2 cell viability, was in the order of PSP-Carb>PSP-Amide>PSP-Imine. Each cationic polymer condensed the luciferase plasmid to nanoparticles of 150–200nm diameters and with a zeta potential of +15–30mV when the mass ratio of polymer-to-DNA was over 8/1. The three polycationic carriers showed similar luciferase transfection activity in COS-7 cells, while the transfection efficiency of PSP-Carb was significantly higher than that of the other two in HepG2 cells. PSP-Amide exhibited significantly higher gene silencing activity in COS-7 cells, suggesting the linkage structures play an important role in the activity of the polyspermine-based nucleic acid carriers.

TIMP-2 gene transfer by positively charged PEG-lated monosized polycationic carrier to smooth muscle cells

Remodeling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes is implicated in restenosis following balloon angioplasty. Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases play an essential role in both normal and pathological extracellular matrix degradation. Tissue inhibitor of matrix metalloproteinase-2 is the most extensively studied tissue inhibitor of metalloproteinases in myocardial tissue in animal models and clinical examples of cardiac disease; therefore it is selected for this study. Gene transfer of tissue inhibitor of matrix metalloproteinase-2 may have a therapeutic potential by inhibition of matrix metalloproteinase activity. We have used PEG-lated nanoparticles poly(St/PEG-EEM/DMAPM) which were synthesized previously in our laboratory. The nanoparticles, with an average size of 77.6 ± 2.05 nm with a zeta potential of +64. 4 ± 1.14 mV and 201.9 ± 1.83 nm with +54.2 ± 0.77 mV were used in the transfection studies. Zeta Potential values and size of polyplex were appropriate for an effective transfection. TIMP-2 expression was detected by western blotting. Increased protein level in smooth muscle cells according to non-transfected smooth muscle cells confirms the successful delivery and expression of the tissue inhibitor of matrix metalloproteinase-2 gene with the non-viral vector transfection approach.

Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier

BackgroundThe reduction-sensitive cationic polymer is a promising nonviral carrier for gene delivery. Until now, disulfide bonds have been the only golden standard for its design. The aim of this research was to develop a novel reduction-responsive cationic polymer as a gene carrier.MethodsPolycationic carriers were synthesized by addition of branched oligoethylenimine 800 Da (OEI800) via an active ester containing diselenide bonds. Disulfide bonds cross-linked with OEI800-SSx and monoselenide bonds linked with OEI800-Sex were synthesized and compared. Their molecular weights and degradation properties were determined using gel permeation chromatography. Changes in particle size, morphology, and DNA binding were investigated by dynamic light scattering, transmission electron microscopy, and electrophoresis assay in a reduction environment. Cytotoxicity and transfection in vitro were evaluated in a murine melanoma cell line (B16F10) and a human cervical epithelial carcinoma cell line (HeLa), while intracellular degradation and dissociation with DNA were studied by confocal laser scanning microscopy with FITC-labeled OEI800 derivatives and Cy5-labeled DNA.ResultsDiselenide-conjugated OEI800 (OEI800-SeSex) polymer carriers of high molecular weight were successfully synthesized. After compacting with DNA, the OEI800-SeSex polymers formed nanoparticles with an average size of 140 nm at an adequate C/P ratio. OEI800-SeSex showed reduction-responsive degradation properties similar to those of the OEI800-SSx via gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. OEI800-SeSex showed much lower cytotoxicity than PEI25k, and significantly higher transfection efficiency than OEI800 in both B16F10 and HeLa cells. Transfection of luciferase in the OEI800-SeSex group was comparable with that of standard PEI25k and traditional reduction-sensitive polymer OEI800-SSx groups. Furthermore, intracellular degradation of OEI800-SeSex and dissociation with DNA were also confirmed by confocal laser scanning microscopy.ConclusionThe OEI800-SeSex obtained was able to bind plasmid DNA efficiently to yield nanosized particles and had reduction sensitivity which is as efficient as that for OEI800-SSx. In vitro experiments confirmed its low cytotoxicity and high transfection ability. Diselenide bonds can be used as effective and novel reduction-sensitive linkages for gene delivery.

Arginine-engrafted biodegradable polymer for the systemic delivery of therapeutic siRNA

Small interfering RNA (siRNA) represent an interesting class of developmental nucleic acid-based therapeutics. Cationic carriers for deoxyribonucleic acids (DNA) are potential vehicles for siRNA delivery. However, in contrast to supercoiled plasmid DNA, the physical properties of siRNA molecules induces the formation of larger, loosely–packed complexes with most polycationic carriers, and consequently, poor target silencing. Here, we investigate a gene delivery agent, arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for siRNA delivery as it contains arginine residues with siRNA binding properties. ABP combines the attributes of polycations and poly disulfide-amines namely- excellent cell-penetrability and rapid release after disulphide bond reduction in the intracellular compartment. ABP bound siRNA, assembled into stable 150 nm sized nanoparticles and efficiently released complexed siRNA upon cellular entry. We investigated the utility of ABP in a combinatorial RNAi strategy for solid cancer therapy. Systemic administration of ABP-siRNA resulted in a preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue. Two administrations of the formulation with a siRNA cocktail targeting Bcl-2, VEGF and Myc at 0.3 mg total siRNA/kg body weight could effectively regress advanced stage tumors. Our results establish the promise of ABP as a common systemic delivery platform for both siRNA and DNA therapeutics.