Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.
ABSTRACT: The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.
Project description:RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.
Project description:In the last 2-3 decades, gene therapy represented a promising option for hepatocellular carcinoma (HCC) treatment. However, the design of safe and efficient gene delivery systems is still one of the major challenges that require solutions. In this study, we demonstrate a versatile method for covalent conjugation of glycyrrhizin acid (GL) or glycyrrhetinic acid (GA) to increase the transfection efficiency of Polyethyleneimine (PEI, Mw 1.8K) and improve their targeting abilities of hepatoma carcinoma cells. GA and GL targeting ligands were grafted to PEI via N-acylation, and we systematically investigated their biophysical properties, cytotoxicity, liver targeting and transfection efficiency, and endocytosis pathway trafficking. PEI-GA0.75, PEI-GL10.62 and PEI-GL20.65 conjugates caused significant increases in gene transfection efficiency and superior selectivity for HepG2 cells, with all three conjugates showing specific recognition of HepG2 cells by the free GA competition assay. The endocytosis inhibition and intracellular trafficking results indicated that PEI-GA0.75 and GL10.62 conjugates behaved similarly to SV40 virus, by proceeding via the caveolae- and clathrin-independent mediated endocytosis pathway and bypassing entry into lysosomes, with an energy independent manner, achieving their high transfection efficiencies. In the HepG2 intraperitoneal tumor model, PEI-GA0.75 and PEI-GL10.62 carrying the luciferase reporter gene gained high gene expression, suggesting potential use for in vivo application.
Project description:Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Noncovalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 kD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 kD PEI polymer were particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in >70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the nontoxic cationic MSNP enhances the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that, by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential.
Project description:Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this paper, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were prepared for the purpose of microRNA (miRNA) delivery. The resultant PEI-AgNCs were characterized by a photoluminescence assay and transmission electron microscopy. A cytotoxicity assay showed that PEI-AgNCs exhibit relatively low cytotoxicity. Interestingly, PEI-AgNCs were confirmed to transfect miRNA mimics more effectively than PEI in HepG2 and 293A cells. In this regard, hsa-miR-21 or hsa-miR-221 mimics (miR-21/221m) were transported into HepG2 cells by using PEI-AgNCs. The miR-21/221 expression was determined post-transfection by quantitative real-time polymerase chain reaction. Compared with the negative control, PEI-AgNCs/miR-21/221m groups exhibited higher miR-21/221 levels. In addition, AgNCs endow PEI with stronger antibacterial activity, and this advantage provided PEI-AgNCs the potential to prevent bacterial contamination during the transfection process. Furthermore, we showed that PEI-AgNCs are viable nanomaterials for plain imaging of the cells by laser scanning confocal microscopy, indicating great potential as an ideal fluorescent probe to track the transfection behavior. These results demonstrated that PEI-AgNCs are promising and novel nonviral vectors for gene delivery.
Project description:Nanoparticles (NPs) can serve as a promising vaccine delivery platform for improving pharmacological property and codelivery of antigens and adjuvants. However, NP-based vaccines are generally associated with complex synthesis and postmodification procedures, which pose technical and manufacturing challenges for tailor-made vaccine production. Here, modularly programmed, polyethyleneimine (PEI)-based NP vaccines are reported for simple production of personalized cancer vaccines. Briefly, PEI is conjugated with neoantigens by facile coupling chemistry, followed by electrostatic assembly with CpG adjuvants, leading to the self-assembly of nontoxic, sub-50 nm PEI NPs. Importantly, PEI NPs promote activation and antigen cross-presentation of antigen-presenting cells and cross-priming of neoantigen-specific CD8<sup>+</sup> T cells. Surprisingly, after only a single intratumoral injection, PEI NPs with optimal PEGylation elicit as high as ?30% neoantigen-specific CD8<sup>+</sup> T cell response in the systemic circulation and sustain elevated CD8<sup>+</sup> T cell response over 3 weeks. PEI-based nanovaccines exert potent antitumor efficacy against pre-established local tumors as well as highly aggressive metastatic tumors. PEI engineering for modular incorporation of neoantigens and adjuvants offers a promising strategy for rapid and facile production of personalized cancer vaccines.
Project description:Three combinatorial libraries of polymeric vectors were evaluated to investigate the functional roles of molecular weight (MW), cations, pH-sensitive moieties, and hydrophobic derivitization in polymer-mediated gene delivery. Four cationic and pH-sensitive moieties (imidazole, primary, secondary, and tertiary amino) and three hydrophobic residues (C4 butyl, C6 hexyl, and C8 octyl) were assessed in single and serially incremented, binary combinations. Three MWs were evaluated-10, 30, and 50 kDa. The highest levels of transfection, comparable to branched PEI (25 kDa), were achieved by 30 kDa and 50 kDa formulations containing primary amino and imidazole groups. Primary amino groups offered superior charge-neutralizing and size-condensing capacity, while imidazole groups appeared to bind with DNA via nonelectrostatically mediated interactions to produce stable polyplexes that were resistant to premature dissociation. Eight of the 10 highest-transfecting polymers possessed IC(50) values greater than the maximum concentration of free polymers exposed to cells (200 microg/ml). The results herein have identified highly efficient polymeric formulations with superb toxicity profiles and have revealed the functional roles that the investigated pendant groups play in the transfection process. The reported polymeric system offers a versatile and robust platform upon which future structure-function studies may be based to create safer and more efficient polymeric vectors.
Project description:Multifunctional nanoparticles as theranostic tools hold great potential for its unique and efficient way to visualize the process of disease treatment. However, the toxicity of conventional fluorescent labels and difficulty of functionalization limit their widespread use. Recently, a number of amino-rich polymers have demonstrated high luminescent fluorescence but rarely showed potential for in vivo imaging due to their blue fluorescence. Here, a general route has been found to construct polymer-based multifunctional nanoparticles for combined imaging and drug delivering. The weak fluorescent polyethyleneimine (PEI) has been conjugated with hydrophobic polylactide as the amphiphilic PEI for construction of nanoparticles which showed bright and multicolor fluorescence with high drug loading capacity. The paclitaxel-loaded nanoparticles showed significant therapy effect in contrast to the free paclitaxel. Meanwhile, fluorescence imaging of the nanoparticles showed accumulation around tumor. These results demonstrate a new type of polymer-based multifunctional nanoparticles for imaging-guided drug delivery.
Project description:An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.
Project description:<h4>Aim</h4>To develop an improved delivery system for nucleic acids.<h4>Materials & methods</h4>We designed, synthesized and characterized a new polymer of lactic-co-glycolic acid-modified polyethylenimine (LGA-PEI). Functions of LGA-PEI polymer were determined.<h4>Results</h4>The new LGA-PEI polymer spontaneously formed nanoparticles (NPs) with DNA or RNA, and showed higher DNA or RNA loading efficiency, higher or comparable transfection efficacy, and lower cytotoxicity in several cell types including PANC-1, Jurkat and HEK293 cells, when compared with lipofectamine 2000, branched or linear PEI (25 kDa). In nude mouse models, LGA-PEI showed higher delivery efficiency of plasmid DNA or miRNA mimic into pancreatic and ovarian xenograft tumors. LGA-PEI/DNA NPs showed much lower toxicity than control PEI NPs in mouse models.<h4>Conclusion</h4>The new LGA-PEI polymer is a safer and more effective system to deliver DNA or RNA than PEI.
Project description:Polyethylenimine (PEI), which is frequently used for polyplex formation and effective gene transfection, is rarely recognized as a luminescent polymer. Therefore, it is usually tagged with an organic fluorophore to be optically tracked. Recently, we developed branched PEI (bPEI) superparamagnetic iron oxide nanoparticles (SPION@bPEI) with blue luminescence 1200 times stronger than that of bPEI without a traditional fluorophore, due to partial PEI oxidation during the synthesis. Here, we demonstrate in vitro dye-free optical imaging and successful gene transfection with luminescent SPION@bPEI, which was further modified for receptor-mediated delivery of the cargo selectively to cancer cell lines overexpressing the epidermal growth factor receptor (EGFR). Pro-apoptotic polyinosinic–polycytidylic acid sodium (PIC) was delivered to HeLa cells with SPION@bPEI and caused a dramatic reduction in the cell viability at otherwise non-toxic nanoparticle concentrations, proving that bPEI coating is still an effective component for the delivery of an anionic cargo. Besides, a strong intracellular optical signal supports the optically traceable nature of these nanoparticles. SPION@bPEI nanoparticles were further conjugated with Erbitux (Erb), which is an anti-EGFR antibody for targeting EGFR-overexpressing cancer cell lines. SPION@bPEI-Erb was used for the delivery of a GFP plasmid wherein the transfection was confirmed by the luminescence of the expressed gene within the transfected cells. Poor GFP expression in MCF7, a slightly better expression in HeLa, and a significant enhancement in the transfection of HCT116 cells proved a selective uptake and hence the targeting ability of Erb-tagged nanoparticles. Altogether, this study proves luminescent, cationic, and small SPION@bPEI nanoparticles as strong candidates for imaging and gene therapy.