•Amphoteric curdlan derivatives were prepared and characterized.•Novel amphoteric curdlan derivatives have negligible cytotoxicity.•Amphoteric curdlan derivatives bind to nucleic acids and form nanoparticles.•Appreciable gene delivery efficiency was achieved by amphoteric curdlan derivatives.•Efficient siRNA delivery by the amphoteric curdlan derivatives was observed in cancer cell lines.Cationic polymers are powerful carriers for intracellular delivery of therapeutic nucleic acids. However, the positively charged macromolecules have considerable cytotoxicity and often induce irreversible damages to the cells and tissues, which greatly negate the clinical application of such materials as drug delivery system. Herein, we report the synthesis of novel amphoteric polymers based on curdlan, and the evaluation of their cytotoxicity as well as the nucleic acid delivery efficiency. β-(1,3)-polyglucuronic acid, a TEMPO-oxidized derivative of curdlan, was chemically modified by conjugation of tetraethylenepentamine. The resulting amphoteric polymers, denoted tetraethylenepentamine-curdlan (TEPAC) polymers have the degree of substitution (DS) ranging from 25% to 48%. The result of MTT assay indicated that TEPAC polymers have negligible cytotoxicity on HeLa cells and A549 cells. The novel amphoteric polymers efficiently bound with plasmid DNA and delivered pcDNA-eGFP plasmid to 293T cells and induced expression of GFP 48 h after the transfection. Moreover, TEPAC polymers delivered siRNA to HeLa cells and HepG2 cells in high efficiency, and induced significant RNAi for the expression of an endogenous gene. Collectively, our data demonstrate that the novel curdlan-based amphoteric polymers are biocompatible and may provide a highly efficient system for the delivery of therapeutic nucleic acids.

RNA interference (RNAi) has shown great potential for clinical treatment of a variety of diseases. As an effective post-transcriptional regulation, RNAi induces degradation of specific target mRNA, leading to a decreased level of disease related gene product, which could be resistant to conventional small molecule therapeutics. Because of the relatively small size of short interfering RNA (siRNA) as well as it's robustness in the design of efficient 20–22 nucleotide sequences targeting any known gene, delivery of siRNA in vitro and in vivo has achieved tremendous success by the design and fabrication of various carrier materials including peptides, lipids, and polymers. While siRNA delivery efficiency of these materials proved to be appreciable, their tissue specificity, biocompatibility and cytotoxicity still need much improvement. We report a novel siRNA delivery material designed by optimization of our previously reported peptidomimetic based lipoplex. The novel lipoplex, which bears a D-amino acid based dipeptide head group, possesses an ideal balance of increased stability in a tissue environment, and enhanced efficiency of tissue specific delivery as well as minimized cytotoxicity. The novel lipoplex carrying siRNA efficiently targeted mouse liver, initiated RNAi and knocked down the apoB dose dependently, which is confirmed by both RT-qPCR and Western blotting. Moreover, the novel lipoplex does not induce any apparent cytotoxicity, as confirmed by the measurement of liver enzyme levels in serum.

Intracellular delivery of genetic material is a potentially powerful therapeutic approach for the treatment of genetic diseases. However, the scarcity of biocompatible delivery systems is a tremendous barrier for the application of nucleic acid therapy in the clinic. Herein, we successfully created biocompatible nucleic acid delivery systems based on cationic lipids consisting of alternatively linked ornithine residues in the head group (denoted DoGo lipids). Transfection of the pcDNA3-eGFP plasmid into cells using DoGo lipids resulted in significant transfection efficiency without showing any apparent cytotoxicity. Meanwhile, DoGo lipids efficiently delivered siRNA to cancer cell lines and induced profound RNAi activity at a siRNA concentration as low as 5 nM. DoGo lipid driven delivery of a siRNA against polo-like kinase 1 (Plk1) in cancer cells induced significant morphological changes in the cell and the nucleus due to cell cycle arrest and inhibition of cell proliferation, a phenotype resulting from the decreased Plk1 protein level. Our findings demonstrate that DoGo lipids have great potential in clinical application for gene based therapy.

RNA interference (RNAi) is a highly efficient approach for gene silencing. Regulation of gene expression at post-transcriptional level provides great potential for curing diseases caused by abnormal overexpression of disease-related genes. However, the application of RNAi in the clinic has been hindered by the lack of efficient and biocompatible delivery systems. Therefore, the development of a safe and tissue-targeted double-stranded interfering RNA (siRNA) carrier for clinical application is urgently needed. Here we report the discovery of a highly efficient liposomal siRNA delivery agent based on a novel peptidomimetic built from natural amino acids. Fine tuning of the composition of amino acids, the type of amide linkage in the peptidomimetic, as well as the formulation and the physicochemical parameters of the novel lipoplex resulted in a lipid nanoparticle (LNP) that efficiently encapsulates and carries siRNA to mouse liver. In vivo experiments showed that a single injection of unmodified siRNA complexed to one of the peptidomimetics at a clinically feasible dose induced significant RNAi in mouse liver, resulting in a 90% decrease in apolipoprotein B (ApoB) mRNA level, as well as a 60% decrease in serum ApoB protein level. Analysis of mouse serum by ELISA indicated that the novel peptidomimetic based lipoplex did not elevate the level of liver enzymes (ALT, AST) in the serum. Our novel peptidomimetic-based lipoplex demonstrated great potential for the development of a safe and efficient siRNA delivery agent for clinical applications.Keywords: ApoB; lipoplex; peptidomimetic; RNAi; siRNA delivery

•6-Amino-6-deoxy-curdlan is PEGylated and characterized.•PEGylated 6-amino-6-deoxy-curdlan shows high affinity to short interfering RNA.•PEGylated 6-amino-6-deoxy-curdlan is biocompatible.•Fluorescein labeled PEGylated 6-amino-6-deoxy-curdlan distributes in mouse liver.•PEGylated 6-amino-6-deoxy-curdlan efficiently delivers siRNA to mouse liver.RNA interference (RNAi) is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies need to establish efficient delivery system that silences target genes with siRNA doses that is clinically feasible in humans. Here we report synthesis and in vivo study of a novel PEGylated curdlan-based nanoparticle, designated as 6AC-100PEG, obtained by conjugation of mPEG 2000 to 6-amino-6-deoxy-curdlan. The complex of siRNA/6AC-100PEG showed homogenous nanoparticles with an average diameter of 200 nm. MTT assay indicated that 6AC-100PEG does not have apparent cytotoxicity. Systemic administration of a complex of siapoB/6AC-100PEG significantly reduced the level of apoB mRNA in mouse liver, indicating that 6AC-100PEG can efficiently deliver siRNA to mouse liver and induce RNAi. Administration of siRNA/6AC-100PEG to mouse did not elevate liver enzyme level in the serum, indicating that 6AC-100PEG nanoparticle is a promising in vivo siRNA delivery agent.

Tissue-specific delivery of therapeutic RNAi has great potential for clinical applications. Receptor-mediated endocytosis plays a crucial role in targeted delivery of biotherapeutics including short interfering RNA (siRNA). Previously we reported a novel Curdlan-based nanoparticle for intracellular delivery of siRNA. Here we designed a nanoparticle based on ligand-functionalized Curdlan. Disaccharides were site-specifically conjugated to 6-deoxy-6-amino Curdlan, and the cell line specificity, cellular uptake, cytotoxicity, and siRNA delivery efficiency of the corresponding disaccharide-modified 6-deoxy-6-amino-Curdlan were investigated. Observation by fluorescence microscopy as well as flow cytometry showed that galactose-containing Curdlan derivatives delivered fluorescently labeled short nucleic acid to HepG2 cells expressing ASGPR receptor but not in other cells lacking surface ASGPR protein. Moreover, highly galactose-substituted Curdlan derivatives delivered siRNA specifically to ASGPR-expressing cells and induced RNAi activities, silencing endogenous GAPDH gene expression. Our data demonstrated that galactose-functionalized 6-deoxy-6-amino-Curdlan is a promising carrier for short therapeutic nucleic acids for clinical applications.Keywords: ASGPR; Curdlan; galactose; receptor-mediated delivery; siRNA delivery

•Water soluble curdlan derivatives were prepared and fully characterized.•The intracellular siRNA delivery efficiency depends on content of 6-amine units.•6-Amino curdlan/siRNA forms nanoparticles.•6-Amino curdlan efficiently delivered siRNA to cancer cells and stem cells.•We report a novel curdlan-based siRNA delivery system.RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70–90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of GFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs.

•“Click” chemistry mediated amine functionalization of curdlan.•Lysine “clicked” curdlan shows excellent water solubility.•Plasmid/Lysine “clicked” curdlan complex forms nanoparticles.•Lysine “clicked” curdlan efficiently delivered eGFP plasmid to cancer cells.•We report a novel curdlan-based gene delivery system.A cationic group has been quantitatively and selectively introduced into C6 position of each glucose units of Curdlan by “Click Chemistry” successfully. The resulting cationic Curdlan-Imidazole-lysine polymers (Cur-6-100Lys) exhibit excellent water solubility. Structure of the Cur-6-100Lys complexes was verified by FTIR and NMR spectroscopic measurements, and analysis of Cur-6-100Lys by GPC, DLS and SEM revealed that they have stoichiometric, nanosized spheroidal structures. Cytotoxicity measurement, electrophoretic mobility shift assay and EGFP-pDNA transfection have been carried out respectively. The results clearly show that Cur-6-100Lys nanocarriers have bound to dsDNA promptly, are less cytotoxic to both 7901 cells and HeLa cells, and are readily able to transport EGFP-pDNA into HepG2 cells. Our studies indicated that Cur-6-100Lys can potentially be used as a versatile nano platform for efficient gene delivery in living cells.

Intracellular delivery of genetic material is a potentially powerful therapeutic approach for the treatment of genetic diseases. However, the scarcity of biocompatible delivery systems is a tremendous barrier for the application of nucleic acid therapy in the clinic. Herein, we successfully created biocompatible nucleic acid delivery systems based on cationic lipids consisting of alternatively linked ornithine residues in the head group (denoted DoGo lipids). Transfection of the pcDNA3-eGFP plasmid into cells using DoGo lipids resulted in significant transfection efficiency without showing any apparent cytotoxicity. Meanwhile, DoGo lipids efficiently delivered siRNA to cancer cell lines and induced profound RNAi activity at a siRNA concentration as low as 5 nM. DoGo lipid driven delivery of a siRNA against polo-like kinase 1 (Plk1) in cancer cells induced significant morphological changes in the cell and the nucleus due to cell cycle arrest and inhibition of cell proliferation, a phenotype resulting from the decreased Plk1 protein level. Our findings demonstrate that DoGo lipids have great potential in clinical application for gene based therapy.