Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Design and Fabrication of Dual Redox Responsive Nanoparticles with Diselenide Linkage Combined Photodynamically to Effectively Enhance Gene Expression.

BACKGROUND: PEI is currently the most used non-viral gene carrier and the transfection efficiency is closely related to the molecular weight; however, the prominent problem is that the cytotoxicity increased with the molecular weight. METHODS: A novel redox responsive biodegradable diselenide cross-linked polymer (dPSP) was designed to enhance gene expression. ICG-pEGFP-TRAIL/dPSP nanoparticles with high drug loading are prepared, which have redox sensitivity and plasmid protection. The transfection efficiency of dPSP nanoparticle was evaluated in vitro. RESULTS: The plasmid was compressed by 100% at the N/P ratio of 16, and the particle size was less than 100 nm. When explored onto high concentrations of GSH/H2O2, dPSP4 degraded into small molecular weight cationic substances with low cytotoxicity rapidly. Singlet oxygen (1O2) was produced when indocyanine green (ICG) was irradiated by near-infrared laser irradiation (NIR) to promote oxidative degradation of dPSP4 nanoparticles. Under the stimulation of NIR 808 and redox agent, the particle size and PDI of ICG-pDNA/dPSP nanoparticle increased significantly. CONCLUSION: Compared with gene therapy alone, co-transportation of dPSP4 nanoparticle with ICG and pEGFP-TRAIL had better antitumor effect. Diselenide-crosslinked polyspermine had a promising prospect on gene delivery and preparation of multifunctional anti-tumor carrier.

Improving Bone Regeneration Using Chordin siRNA Delivered by pH-Responsive and Non-Toxic Polyspermine Imidazole-4,5-Imine.

BACKGROUND/AIMS: Bone nonunion remains a challenge for orthopaedists. The technological advancements that have been made in precisely silencing target genes have provided promising methods to address this challenge. METHODS: We detected the expression levels of the bone morphogenetic protein (BMP) inhibitors Chordin, Gremlin and Noggin using realtime PCR in bone mesenchymal stem cells (BMSCs) isolated from patients with normal fracture healing and those with bone nonunion. Moreover, we detected the expression of Chordin, Gremlin and Noggin during the osteogenic differentiation of human BMSCs (hBMSCs) using real-time PCR and Western blot. We delivered Chordin siRNA to hBMSCs using a previously reported cationic polymer, polyspermine imidazole-4,5-imine (PSI), as a pH-responsive and non-cytotoxic transfection agent. The apoptosis and cellular uptake efficiency were analysed by flow cytometry. RESULTS: We identified Chordin as the most appropriate potential therapeutic target gene for enhancing the osteogenic differentiation of hBMSCs. Chordin knockdown rescued the osteogenic capacity of hBMSCs isolated from patients with bone nonunion. Highly efficient knockdown of Chordin was achieved in hBMSCs using PSI. Chordin knockdown promoted hBMSC osteogenesis and bone regeneration in vitro and in vivo. CONCLUSIONS: Our results suggest that Chordin is a potential target for improving osteogenesis and bone nonunion therapy and that responsive and non-toxic cationic polyimines such as PSI are therapeutically feasible carriers for the packaging and delivery of Chordin siRNA to hBMSCs.

A targeted nanocarrier based on polyspermine for the effective delivery of methotrexate in nasopharyngeal carcinoma.

Antitumor efficacy of methotrexate (MTX) is seriously limited due to its poor water solubility, nonspecific tumor distribution and low bioavailability. To overcome these obstacles, polyspermine (PSP) conjugated with MTX and folic acid (FA) as a novel targeted prodrug was designed and has been successfully synthesized using the amidation reaction. The strong hydrophilic properties of PSP made MTX well dispersed in water and the cellular uptake study indicated that the presence of FA enhanced uptake of the FA-PSP-MTX in folate receptor (FR) over-expressing human nasopharyngeal carcinoma HNE-1 cells. 1H NMR spectra and UV-Vis spectral analysis were carried out to confirm the MTX and FA content in FA-PSP-MTX, respectively. In CCK-8 assay and apoptosis analysis, the prodrug showed significantly enhanced anticancer efficacy than free MTX in HNE-1 cells. These results suggested that the prodrug has the potential for targeted delivery of MTX into cancer cells to improve its anti-tumor efficacy.

Folate-conjugated polyspermine for lung cancer-targeted gene therapy.

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamine(®) 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.

Folate-conjugated polyspermine for lung cancer-targeted gene therapy

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamine(®) 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.

A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy.

The clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 µg/mL at different time (24 and 72 h), which much higher than PEI 25K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras(LA1) lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy.

Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy.

The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD-SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results demonstrate that SD-SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy.

Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites.

Cationic polymers readily degradable in response to cellular environment are especially favored as easy-formulating materials to pack siRNA into a nanoparticle and to release the cargo in the cytoplasm in time. In addition to the efficiency of cytosomal release, the degradation products should best be free of safety concerns, a typical challenge for cationic polymers. To satisfy the two criteria, we report a new cationic polymer, polyspermine imine, named as PSP-Imine, which is formed by condensing two endogenous molecules, spermine and glyoxal, through conjugated π linkage, -N═C-C═N- (Schiff base reaction), a poly linkage structure sufficiently stable under neutral condition but dissociative under the endosomal pH. Cellular assays under a confocal microscope indicated that the polyplex formed of PSP-Imine readily released the loaded siRNA to the cytoplasm after being engulfed in the target cells and efficiently silenced the target genes in different cell lines and xenograft mouse model of human cervical carcinoma, as compared with nondegradable PEI 25 kDa. Cell viability assays confirmed that PSP-Imine showed no visible cytotoxicity within the concentration being tested. The present study suggests that PSP-Imine is an excellent siRNA condensing material for forming the core of a therapeutically feasible synthetic carrier system.

Spermine-alt-poly(ethylene glycol) polyspermine as a safe and efficient aerosol gene carrier for lung cancer therapy.

The clinical success of gene therapy critically depends upon the safety and efficiency of delivery system used. Although polyethylenimine (PEI) has been commonly used as an efficient cationic polymeric gene carrier due to its high transfection efficiency, its cytotoxicity and nondegradability limit the polymer's therapeutic applications in clinical trials. In this study, biocompatible polyspermine based on spermine (SPE) and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) was synthesized using a Michael-type addition reaction, and its ability as an alternative gene carrier for lung cancer therapy was evaluated. SPE-alt-PEG polyspermine was complexed with plasmid DNA, and the resulting complexes were characterized by particle size and surface charge by dynamic light scattering, complex formation and DNA protection ability by gel retardation, and complex shape by energy-filtering transmission electron microscopy. The SPE-alt-PEG copolymer showed low cytotoxicity, and SPE-alt-PEG/DNA complexes showed efficacious transfection efficiency compared with 25 kDa PEI (PEI 25K). Also SPE-alt-PEG/GFP complexes were efficiently transferred into the lungs after aerosol administration without toxicity, and delivery of Pdcd4 gene as a therapeutic gene with SPE-alt-PEG polyspermine greatly reduced tumor size as well as tumor numbers in K-ras(LA1) lung cancer model mice compared relative to the effect observed for PEI 25K. These results suggest that SPE-alt-PEG has potential as a gene carrier for lung cancer gene therapy.

Synthetic polyspermine imidazole-4, 5-amide as an efficient and cytotoxicity-free gene delivery system.

A chemically dynamic spermine-based polymer: polyspermine imidazole-4, 5-amide (PSIA, Mw > 7 kDa) was designed, synthesized, and evaluated in terms of its ability to deliver nucleic acids. This polymer was made from an endogenous monomer professionally condensing genes in sperms, spermine, and a known safety drug metabolite, imidazole-4, 5-dicarboxylic acid, through a bis-amide bond conjugated with the imidazole ring. This polymer can condense pDNA at a W/W ratio above 10 to form polyplexes (100-200 nm in diameter), which is consistent with the observation by transmission electron microscopy (TEM), and the zeta potential was in the range of 10-20 mV. The pDNA packaged polymer was stable in phosphate buffer solution (PBS) at pH 7.4 (simulated body fluid) while the polyplexes were releasing pDNA into the solution at pH 5.8 (simulated endo-lysosomes) due to the degradation of the bis-amide linkages in response to changes in pH values. PSIA-polyplexes were able to achieve efficient cellular uptake and luciferase gene silencing by co-transfection of pDNA and siRNA in COS-7 cells and HepG2 cells with negligible cytotoxicity. Biodistribution of Rhodamine B-labeled PSIA-polyplexes after being systemically injected in BALB/c nude-mice showed that the polyplexes circulated throughout the body, accumulated mainly in the kidney at 4 hours of sample administration, and moved to the liver and spleen after 24 hours. All the results suggested that PSIA offered a promising example to balance the transfection efficiency and toxicity of a synthetic carrier system for the delivery of therapeutic nucleic acids.