Light-Controlled in Vitro Gene Delivery Using Polymer-Tethered Spiropyran as a Photoswitchable Photosensitizer.

A gene delivery system using spiropyran as a photoswitchable photosensitizer for the controlled photochemical internalization effect was developed by engineering the outer coating of a polyethylenimine/DNA complex with a small amount of spiropyran-containing cationic copolymers. The successful binding of cationic polymers by the polyethylenimine coating was detected by the distance-sensitive fluorescence resonance energy-transfer technique that evidenced the occurrence of energy transfer between fluorescein-labeled cationic copolymers and polyethylenimine-condensed rhodamine-labeled DNA. The ternary polyplexes feature reversible controllability of singlet oxygen generation based on the dual effect of spiropyrans in photochromism and aggregation-induced enhanced photosensitization, allowing significant light-induced amplification of bPEI-mediated in vitro transgene efficiency (from original 15% to final 91%) at a low DNA dose, with the integrity of supercoiled DNA structure unaffected. The use of spiropyran without the need of other photosensitizers circumvents the issue of uncontrolled long-lasting photocytotoxicity in gene delivery.

Spiropyran in nanoassemblies as a photosensitizer for photoswitchable ROS generation in living cells.

Reversibly controlled generation of singlet oxygen from photosensitizing nanosystems has the benefits of selective cell killing and controllable effect time, but is a challenging option for photodynamic therapies. We report a strategy for integrating photochromic spiropyrans into biocompatible cationic polymers, which involved assembling nucleic acids into functional nanoparticles without introducing additional photosensitizers and imaging agents. We found that spiropyran-containing nanoparticles have photoswitching properties for both fluorescence (with a quantum yield of up to 0.27) and singlet oxygen generation (with a quantum yield of up to 0.22) in aqueous solutions and cells, and demonstrated that spiropyrans in nanoassemblies featuring aggregation-induced enhanced photosensitization and emission could be potentially applied in photodynamic therapy studies on tumor cells.

Remarkable Amplification of Polyethylenimine-Mediated Gene Delivery Using Cationic Poly(phenylene ethynylene)s as Photosensitizers.

Conjugated polymers can serve as good photosensitizers in biomedical applications. However, it remains unknown whether they are phototoxic to the supercoiled structure of DNA in improving gene delivery by the photochemical internalization (PCI) strategy, which complicates the application of conjugated polymers in gene delivery. In this work, we introduced a trace amount of cationic poly(phenylene ethynylene)s (cPPEs) into the polymeric shell of branched polyethylenimine (BPEI)/DNA complexes, studied the photosensitization of singlet oxygen by cPPEs, and confirmed that the supercoiled DNA is undamaged by the singlet oxygen generated by the photoexcitation of cPPEs. By taking advantage of the cPPE-mediated PCI effect, we report that the addition of the trace amount of cPPEs to the outer shell of the BPEI/DNA polyplexes could greatly amplify the transfection of gene green fluorescent protein on tumor cells with the efficiency from 14 to 86% without decreasing the cell viabilities, well solving the problem with a poor transfection capability of BPEI under low DNA-loading conditions. Our strategy to employ conjugated polymers as photosensitizing agents in gene delivery systems is simple, safe, efficient, and promising for broad applications in gene delivery areas.

Facile crosslinking of polythiophenes by polyethylenimine via ester aminolysis for selective Cu(II) detection in water.

Functionalization of π-conjugated polymers is dispensable for solubilization of the rigid and hydrophobic backbones in water. However, polymer aggregation is always present and leads to issues with complication and reproducibility in spectral properties. Herein, we reported a simple and robust method to make a series of conjugated polymer nanostructures by a crosslinking strategy. In favor of multivalency effect, polythionenes with various chain lengths were functionalized with branched polyethylenimine (PEI) via ester aminolysis reaction under mild conditions. Photophysical studies revealed the conjugated backbones could be well stabilized and dispersed in water. By taking advantage of intermolecular recognition interaction between copper ions and cationic PEI, we applied crosslinked polythiophenes as a nano probe at very low concentration (0.01 mg/mL) to fluorescently detect copper ions with high sensitivity up to 10 nM and selectivity over other metal ions in aqueous solutions, without occurrence of detectable aggregates. The overall performance of our nano probes outperforms reported water-soluble polymers-based probes, particularly in probe availability and manipulation as well as selective copper detection capability.

Synthesis of Structurally Defined Cationic Polythiophenes for DNA Binding and Gene Delivery.

Water-soluble conjugated polymers (WCPs) have prospective applications in the field of bioimaging, disease diagnosis, and therapy. However, the use of WCPs with controllability and regioregularity for bioapplications have scarcely been reported. In this work, we synthesized polythiophenes containing ester side chains (P3ET) via Kumada catalyst-transfer polycondensation (KCTP) and confirmed a quasi-"living" chain-growth mechanism. In addition, we obtained cationic regioregular polythiophenes (cPTs) by aminolysis of P3ET with varied chain lengths, and studied DNA binding capability and gene delivery performance. Benefiting from photocontrolled generation of intracellular reactive oxygen species (ROS), the cationic polythiophenes successfully delivered DNA into tumor cells without additional polymer species.

Galactosylated poly(ethylene glycol) methacrylate-st-3-guanidinopropyl methacrylamide copolymers as siRNA carriers for inhibiting Survivin expression in vitro and in vivo.

In this report, galactosylated poly(ethylene glycol) methacrylate-st-3-guanidinopropyl methacrylamide copolymers (galactosylated PEGMA-st-GPMA, GGP) are developed as siRNA carriers to inhibit Survivin mRNA expression. GGPs are combined with Survivin siRNAs to form siRNA/GGP polyplexes. The polyplexes particles were examined by a dynamic light scattering. It showed that GGP copolymers could condense siRNA to form particles with diameter from 128 to 423 nm and zeta potential value in the range from +2.4 to +14.9 mV at various charge ratios (N/P). The MTT assay data of siRNA/GGP polyplexes on human hepatocellular liver carcinoma cells (HepG2) and human cervix epithelial carcinoma cells (HeLa) indicated that GGP copolymer had better cell viabilities than polyethyleimine (PEI). The transfection of siRNA/GGP polyplexes was detected by real-time quantitative PCR (RT-qPCR) in HepG2 cell line. We found that the siRNA/GGP polyplexes could effectively silence Survivin mRNA expression in the serum-free media (p < 0.01). In the presence of 10% serum medium, the Survivin mRNA expressed has significant difference between siRNA/GGP polyplexes and blank (p < 0.05). The galactose competition assay showed that galactosylated PEGMA-st-GPMA (GGP) may provide the targeting to HepG2 cells mediating by asialoglycoproteins receptors (ASGP-R). Furthermore, Survivin siRNA/GGP polyplexes could significantly (p < 0.01) inhibit both HepG2 tumor growth and Survivin protein expression in vivo studies in a xenograft mouse model.

Guanidinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymer as siRNA carriers for inhibiting human telomerase reverse transcriptase expression.

In this report, a series of well-defined glucose- and guanidine-based cationic copolymers as gene carriers were developed to inhibit human telomerase reverse transcriptase (hTERT) gene expression. First of all, guandinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymers (guanidinylated GAPMA-s-APMA, abbreviated as GGA) were prepared via aqueous reversible addition--fragmentation chain transfer polymerization (RAFT). Then, three target hTERT siRNA TERT-1, TERT-2 and TERT-3 were designed and combined with GGA copolymers to form siRNA/GGA polyplexes. The polyplexes were examined by dynamic light scattering and agarose gel electrophoresis. The results indicated that GGA copolymers can condense siRNA effectively to form particles with the diameter from 157 nm to 411 nm and zeta potential values in the range from +3.7 to +15.8 mV at various charge ratios (N/P). The MTT assay data of siRNA/GGA polyplexes on human hepatocellular liver carcinoma cells (HepG2) indicated that GGA copolymer had better cell viabilities than polyethylenimine (PEI). Furthermore, the transfection of siRNA/GGA polyplexes was detected by real-time quantitative PCR (RT-qPCR) in HepG2. It was found that siRNA/GGA polyplexes could effectively silence hTERT mRNA expression in serum-free media (p<0.01). In the presence of serum, the hTERT mRNA expression in HepG2 cells have significant difference (p<0.01) between siRNA/GGA3 polyplexes and blank. The results showed that the GAPMA component can reduce the aggregation of protein in serum media. Therefore, the enhancement of transfection may be attributed to the combination of guadino groups and glucose component. And, the guandinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymers might be promise in gene delivery.