交联型聚乙烯亚胺智能基因载体的制备及PEG化影响

使用胱胺双丙烯酰胺(CBA)对低分子量聚乙烯亚胺(PEI)进行交联反应制备智能降解型聚阳离子基因载体.通过与聚乙二醇(PEG)反应得到不同程度PEG化的聚阳离子载体.利用核磁、黏度测试、粒度仪、zeta电位仪和凝胶电泳对聚阳离子载体及其与DNA的复合物进行了表征.研究表明随着PEG含量的增加,聚阳离子载体/DNA复合物颗粒粒径变小、表面正电荷降低,PEG具有明显的屏蔽作用,但过多的PEG也使载体与DNA复合能力下降.通过MTT细胞毒性测试和荧光素酶质粒转染实验得出,含二硫键的交联型阳离子聚合物在测试范围内显示了非常低的细胞毒性,最佳转染效率是PEI25k的4倍,PEG化后其细胞毒性得到进一步改善,转染效率却明显降低.

SYNTHESIS AND CHARACTERIZATION OF A CROSSLINKING POLYETHYLENIMINE AS SMART GENE CARRIER AND EFFECTS OF PEGYLATION DEGREE

A smart biodegradable cationic polymer (CBA-PEI) based on the disulfide bond-containing cross-linker cystamine bisacrylamide (CBA) and low molecular weight branched polyethylenimine (1800-Da, PEI1800) was successfully synthesized by Michael addition reaction in our recent study. Furthermore, a series of copolymers (CBA-PEI-PEG) with different PEGylation degree were obtained by the mPEG-SPA (5000-Da) reacting with CBA-PEI at various weight ratios directly. The molecular structures of the resulting polymers CBA-PEI and CBA-PEI-PEG were evaluated by nuclear magnetic resonance spectroscopy (H-1-NMR) and capillary viscosity measurements, all of which had successfully verified formation of the copolymers. The polymer/DNA complexes based on CBA-PEI and CBA-PEI-PEG were measured by dynamic light scattering and gel retardation assay. The results showed that the particle size and zeta potential of complexes were reduced with increasing amount of PEG grafting, even no particle formation. The particle size of CBA-PEI/DNA complexes was in range of 103.1 to 129.1 nm, and the zeta potential was in range of 14.2 to 24.3 mV above the 2:1 weight ratio. In the same measure condition, the particle size of CBA-PEI-PEG complexes was reduced to a range of 32.2 to 55 nm, and the zeta potential was in range of 9.3 to 13.8 mV at the 2:1 weight ratio.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Multi-armed poly(L-glutamic acid)-graft-oligoethylenimine copolymers as efficient nonviral gene delivery vectors

Background The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low-toxic, highly efficient gene delivery.Methods A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi-armed poly(L-glutamic acid) backbone. The molecular structures of multi-armed poly(L-glutamic acid)-graft-OEI (MP-g-OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP-g-OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP-g-OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney-293 cells for their cytotoxicity and transfection efficiency.

A general route to prepare one- and three-dimensional carbon nanotube/metal nanoparticle composite nanostructures

Adsorption of polyethyleneimine (PEI)-metal ion complexes onto the surfaces of carbon nanotubes (CNTs) and subsequent reduction of the metal ion leads to the fabrication of one-dimensional CNT/metal nanoparticle (CNT/M NP) heterogeneous nanostructures. Alternating adsorption of PEI-metal ion complexes and CNTs on substrates results in the formation of multilayered CNT films. After exposing the films to NaBH4, three-dimensional CNT composite films embedded with metal nanoparticles (NPs) are obtained. UV-visible spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy are used to characterize the film assembly. The resulting (CNT/M NP)(n) films inherit the properties from both the metal NPs and CNTs that exhibit unique performance in surface-enhanced Raman scattering (SERS) and electrocatalytic activities to the reduction of O-2; as a result, they are more attractive compared to (CNT/polyelectrolyte)(n) and (NP/polyelectrolyte)(n) films because of their multifunctionality.

Effect of main-chain rigidity on the phase transitional behavior of comblike polymers

The influence of the rigidity of polymer backbones on the side-chain crystallization and phase transition behavior was systematically investigated by a combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), Fourier transform infrared spectroscopy (FTIR), and high-resolution solid-state nuclear magnetic resonance spectroscopy (NMR). DSC investigation indicated that the crystallization number of alkyl carbon atoms of the side chains grafted onto the rigid polymer backbone, poly(p-benzamide) (PBA), is much lower than that of the alkyl carbon atoms of the side chains grafted onto the flexible polymer backbone, poly(ethyleneimine) (PEI), implying that the conformational state of the polymer backbones has a strong effect on the side-chain crystallization behavior in comblike polymers. WAXD and FTIR results proved that these two comblike polymers pack into hexagonal (PBA18C) and orthorhombic (PEI18C) crystals, respectively, depending on the adjusting ability of the polymer backbones for particular conformational states. It was also found that the presence of the crystalline-amorphous interphase (delta = 31.6 ppm) in PBA18C detected by solid-state C-13 NMR spectroscopy can be attributed to the rigid PBA backbone, which restricts the mobility of the alkyl side chains.

Order-disorder transition in eicosylated polyethyleneimine comblike polymers

Order-disorder transition (ODT) behavior in eicosylated polyethyleneimine (PEI20C) comblike polymer obtained by grafting n-eicosyl group on polyethyleneimine backbone was systematically investigated by the combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR) spectroscopy as well as solid-state high resolution nuclear magnetic resonance (NMR) spectroscopy. DSC investigations showed two obvious transitions, assigned to the transitions (1) from orthorhombic to hexagonal and (2) from hexagonal to amorphous phase, respectively. These transitions are induced by the variations of alkyl side chain conformation and packing structure with temperature changing, which consequently lead to the destruction of original phase equilibrium. The ODT behavior can also be confirmed by spectroscopic methods like WAXD, FTIR and NMR. The ordered structure and the transition behavior of the alkyl side chains confined by the PEI backbone are obviously different from those of pristine normal alkanes. The transition mechanism of ODT and the origin of the phase transition behavior in PEI20C comblike polymer were discussed in detail in this paper.

Achiral lanthanide alkyl complexes bearing N,O multidentate ligands. Synthesis and catalysis of highly heteroselective ring-opening polymerization of rac-lactide

Alkane elimination reactions of amino-amino-bis(phenols) H2L1-4, Salan H2L5, and methoxy-beta-diimines HL6,7 with lanthanide tris(alkyl) s, Ln(CH2SiMe3)(3)(THF)(2) (Ln = Y, Lu), respectively, afforded a series of lanthanide alkyl complexes 1-8 with the release of tetramethylsilane. Complexes 1-6 are THF-solvated mono( alkyl) s stabilized by O, N, N, O-tetradentate ligands. Complexes 1-3 and 5 adopt twisted octahedral geometry, whereas 4 contains a tetragonal bipyramidal core. Bearing a monoanionic moiety L-6 (L-7), complex 7 ( 8) is a THF-free bis(alkyl). In complex 7, the O, N, N-tridentate ligand combined with two alkyl species forms a tetrahedral coordination core. Complexes 1, 2, and 3 displayed modest activity but high stereoselectivity for the polymerization of rac-lactide to give heterotactic polylactide with the racemic enchainment of monomer units P-r ranging from 0.95 to 0.99, the highest value reached to date. Complex 5 exhibited almost the same level of activity albeit with relatively low selectivity. In contrast, dramatic decreases in activity and stereoselectivity were found for complex 4. The Salan yttrium alkyl complex 6 was active but nonselective. Bis(alkyl) complexes 7 and 8 were more active than 1-3 toward polymerization of rac-LA, however, to afford atactic polylactides due to di-active sites. The ligand framework, especially the "bridge" between the two nitrogen atoms, played a significant role in governing the selectivity of the corresponding complexes via changing the geometry of the metal center.

Metabolic profiling of serum from gadolinium chloride-treated rats by H-1 NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl3, 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168 h postdose (p.d.) after exposure to GdCl3. H-1 NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl3 treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl3 was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl3 in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.

Mn(II)-monosubstituted polyoxometalates as candidates for contrast agents in magnetic resonance imaging

Two mono-substituted manganese polyoxometalates, K6MnSiW11O39 (MnSiW11) and K8MnP2W17O61 (MnP2W17), have been evaluated by in vivo and in vitro experiments as the candidates of potential tissue-specific contrast agents for magnetic resonance imaging (MRI). T-1-relaxivities of 12.1 mM(-1) s(-1) for MnSiW11 and 4.7 mM(-1) s(-1) for MnP2W17 (400 MHz, 25 degrees C) were higher than or similar to that of the commercial MRI contrast agent (GdDTPA). Their relaxivities in BSA and hTf solutions were also reported. After administration of MnSiW11 and MnP2W17 to Wistar rats, MR imaging showed longer and remarkable enhancement in rat liver and favorable renal excretion capability. The signal intensity increased by 74.0 +/- 4.9% for the liver during the whole imaging period (90 min) and by 67.2 +/- 5.3% for kidney within 20-70 min after injection at 40 +/- 3 mu mol kg(-1) dose for MnSiW11. MnP2W17 induced 71.5 +/- 15.1%. enhancement for the liver in 10-45 min range and 73.1 +/- 3.2% enhancement for kidney within 5-40 min after injection at 39 +/- 3 mu mol kg(-1) dose. In vitro and in vivo study showed MnSiW11 and MnP2W17 being favorable candidates as the tissue-specific contrast agents for MRI.

The gadolinium complexes with polyoxometalates as potential MRI contrast agents

The two gadolinium (Gd) polyoxometalates, K-15[Gd(BW11O39)(2)] [Gd(BW11)(2)] and K-17[Gd(CuW11O39)(2)] [Gd(CuW11)(2)] have been evaluated by in vivo and in vitro experiments as the candidates of potential tissue-specific magnetic resonance imaging (MRI) contrast agents. T-1 relaxivities of 17.12 mM(-1) . s(-1) for Gd(BW11)(2) and 19.95 mM(-1) . s(-1) for Gd(CuW11)(2) (400MHz, 25 degrees C) were much higher than that of the commercial MRI contrast agent (GdDTPA). Their relaxivities in bovine serum albumin and human serum transferrin solutions were also reported. After administration of Gd(BW11)(2) and Gd(CuW11)(2) to Wistar rats, MRI showed longer and remarkable enhancement in rat liver and favorable renal excretion capability. The signal intensity increased by 37.63 +/- 3.45% for the liver during the whole imaging period (100 min) and by 61.47 +/- 10.03% for kidney within 5-40 min after injection at 40 +/- 1-mu mol . kg(-1) dose for Gd(CuW11)(2), and Gd(BW11)(2) induced 50.44 +/- 3.51% enhancement in the liver in 5-50-min range and 61.47 +/- 10.03% enhancement for kidney within 5-40 min after injection at 39 +/- 4 mu mol . kg(-1) dose. In vitro and in vivo study showed that Gd(BW11)(2) and Gd(CuW11)(2) are favorable candidates as tissue-specific contrast agents for MRI.

Phase transition and conformational variation of N-alkylated branched poly(ethyleneimine) comblike polymer

A series of branched poly(ethyleneimine) (PEI) derived polymers with different lengths of n-alkyl side chains, denoted as PEI(n)Cs (n = 12, 14, 16, 18, 20, number of carbon atoms in alkyl side group), have been prepared by a N-alkylation method, and systematically characterized by differential scanning calorimertry (DSC) and wide-angle X-ray diffraction (WARD) as well as Fourier transform infrared spectroscopy (FTIR). The side chains grafted on these comblike polymers are long enough to form crystalline phase composed of paraffin-like crystallites. The crystallization of the side chains forces the branched poly(ethyleneimine) molecules to pack into layered structure, between which the crystallites are located. The melting temperatures of the side chain crystallites increase from -12.36 to +51.49 degreesC with increasing the length of the side chains from n. = 12 to n = 20, which are a little bit lower than the corresponding pristine n-alkanes. PEI18C was taken as an example in this work for the investigation of phase transition and conformational variation of the side chains with temperature changing.

NMR and pattern recognition studies on the acute biochemical effects of Lu(NO3)(3)

Pattern recognition methods were applied to the analysis of 600 MHz H-1 NMR spectra of urine from rats dosed with compounds that induced organ-specific damage in the liver and kidney. Male Wistar rats were separated into groups (n=4) and each was treated with one of following compounds: HgCl2, CCl4, Lu(NO3)(3) and Changle (a kind of rare earth complex mixed with La, Ce, Pr and Nd). Urine samples from the rats dosed with HgCl2, CCl4 and Lu(NO3)(3) were collected over a 24 h time course and the samples from the rats administrated with Changle were gained after 3 months. These samples were measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed to provide 223 intensity-related descriptors of spectra. Urine spectral data corresponding to the time intervals, 0-8 h (HgCl2 and CCl4), 4-8 (Lu(NO3)(3)) h and 90 d (Changle) were analyzed using principal component analysis (PCA). Successful classification of the toxicity and biochemical effects of Lu(NO3)(3) was achieved.

Investigation of sandwiched gadolinium(III) complexes with tungstosilicates as potential MRI contrast agents

Two gadolinium-sandwiched complexes with tungstosilicates, K-13[Gd(SiW11O39)(2)] (Gd(SiW11)(2)) and K11H6[Gd2O3(SiW9O34)(2)] (Gd-3(SiW9)(2)), have been investigated by in vitro and in vivo experiments as potential contrast agents for magnetic resonance imaging (MRI). T-1-relaxivity of Gd(SiW11)(2)was 6.59 mM(-1) . s(-1) in aqueous solution and 6.85 mM(-1) . s(-1) in 0.725 mmol . L-1 bovine serum albumin solution at 25degreesC and 9.39 T, respectively. The corresponding T-1-relaxivity of Gd-3(SiW9)(2) was 12.6 and 19.3 mM(-1) . s(-1) per Gd, respectively. MRI for Sprague-Dawley rats showed longer and more remarkable enhancement in rat liver after i.v. injection of these two complexes: 39.4 +/- 3.9% and 57.4 +/- 11.6% within the first 30 min after injection, 31.2 +/- 2.6% and 39.9 +/- 7.6% in the next 60 min for Gd(SiW11)(2) and Gd-3(SiW9)(2) at doses of 0.081 and 0.084 mmol Gd/kg, respectively. Our preliminary in vitro and in vivo study indicates that Gd(SiW11)(2) and Gd-3(SiW9)(2) are favorable candidates for hepatic contrast agents for MRI. However, the two complexes exhibit higher acute toxicity and need to be modified and studied further before clinical use.

Gadolinium heteropoly complex K-17[Gd(P2W17O61)(2)] as a potential MRI contrast agent

Gadolinium heteropoly complex K-17[Gd(P2W17O61)(2)] has been evaluated by in vitro and in vivo experiments as a potential contrast agent for magnetic resonance imaging (MRI). The thermal analysis and conductivity study indicate that this complex has good thermal stability and wide pH stability range. The T-1 relaxivity is 7.59 mM(-1) s(-1) in aqueous solution and 7.97 mM(-1) s(-1) in 0.725 mmol l(-1) bovine serum albumin (BSA) solution at 25degreesC and 9.39 T, respectively. MR imaging of three male Sprague-Dawley rats showed remarkable enhancement in rat liver after intravenous injection, which persisted longer than with Gd-DTPA. The signal intensity increased by 57.1 +/- 16.9% during the whole imaging period at 0.082 mmol kg(-1) dose. Our preliminary in vitro and in vivo studies indicate that K-17[Gd(P2W17O61)(2)] is a potential liver-specific MRI contrast agent.