Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

Aim： To study polyethylenimine （PEI）-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary spermatocytes）. Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. Conclusion： PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.

Nischarin-siRNA delivered by polyethyleniminealginate nanoparticles accelerates motor function recovery after spinal cord injury

A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2 a cells and primary cortical neurons.In recent years,more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury.We proposed that small interfering RNA targeting nischarin（Nis-si RNA） delivered by polyethyleneimine-alginate（PEIALG） nanoparticles promoted motor function recovery in rats with spinal cord injury.Direct microinjection of 5 μL PEI-ALG/Nis-si RNA into the spinal cord lesion area of spinal cord injury rats was performed.From day 7 after surgery,Basso,Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-si RNA group compared with the spinal cord injury group and PEI-ALG/Control-si RNA group.On day 21 after injection,hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-si RNA group.Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-si RNA group.These findings suggest that a complex of PEI-ALG nanoparticles and Nis-si RNA effectively suppresses nischarin expression,induces expression of growth-associated protein-43,and accelerates motor function recovery after spinal cord injury.

Synthesis and Characterization of Poly (Methyl Methacrylate)/Polyethylenimine Grafting Core-Shell Nanoparticles for CO2 Adsorption Using Soap-Free Emulsion Copolymerization

Unlike previous emulsion polymerization, we used grafting reactions in soap-free emulsion systems. In this study, we synthesized grafted PMMA/PEI core-shell nanoparticles by varying the MMA/PEI content and molecular weight of PEI (Mn = 600, 8000, and 10,000). The size and morphology of the core-shell nanoparticles were characterized by a particle size analyzer and scanning electron microscopy. The nanoparticles were 178 - 408 nm in diameter and swelled in water or methanol by 30 - 75 nm. The size of the nanoparticles increased with MMA contents, whereas the size distribution progressively became homogeneous with increasing molecular weight of PEI. Lastly, we measured CO2 adsorption capacity of the grafted PMMA/PEI core-shell nanoparticles, and we found the capacity to be limited at a level of 0.69 mg, which occurred for nanoparticles prepared from emulsions at a pH value of 11.

Surface treatment on polyethylenimine interlayer to improve inverted OLED performance

Polyethylenimine(PEI) interlayer rinsing with different solvents for inverted organic light emitting diodes(OLEDs)is systematically studied in this paper. In comparison with the pristine one, the maximum current efficiency(CE) and power efficiency(PE) are enhanced by 21% and 22% for the device rinsing by ethylene glycol monomethyl ether(EEA).Little effect is found on the work function of the PEI interlayer rinsed by deionized water(DI), ethanol(EtOH), and EEA.On the other hand, the surface morphologies of PEI through different solvent treatments are quite different. Our results indicates that the surface morphology is the key to improving the device performance for IOLED as the work function of PEI keeps stable.

Effect of Polyethylenimine Content on Liposome/Polyethylenimine Complexes-Mediated Gene Delivery

Cationic liposome(Lipo) and polyethylenimine(PEI) are widely applied for nonviral gene transfection.In this study,in order to combine the favorable properties of Lipo and PEI systems for gene delivery,Lipo/PEI complexes with different contents of PEI(5%,10%,20% and 40% relative to phosphatidyl choline in reaction system) were prepared.The physicochemical properties of Lipo/PEI complexes,as well as the influences of PEI content on the storage stability,cytotoxicity and transfection efficiency were investigated.The transmission electron microscopy(TEM) images showed that Lipo/PEI complexes had smaller size compared to pure Lipo.The zeta potential values decreased with the increasing content of PEI.After storaged for 3 months at 4 ℃,obvious aggregation was observed when the addition of PEI content was up to 20%.In vitro cytotoxicity assay showed that Lipo/PEI complexes had decreased cytotoxicity over pure PEI,while the cytotoxicity was enhanced as the PEI content increased.Importantly,the luciferase activity assay and confocal microscope observation revealed that Lipo/PEI complexes prepared with the lowest amount of PEI(Lipo/PEI-5%)possessed the highest transfection efficiency.Thus,these results suggest that feeding the appropriate content of PEI in Lipo/PEI complexes allows them to be excellent vehicle for gene delivery.

Utilization of Polyethylenimine(PEI)Modified Carbon Black Adsorbent Derived from Tire Waste for the Removal of Aspirin

A new adsorbent was synthesized using polyethylenimine(PEI)on the carbon black to remove aspirin from an aqueous solution.In this study,adsorption performance of modified carbon black by polyethylenimine(PEI)on aspirin was investigated.Batch adsorption studies were performed to evaluate the effects of contact time,pH solution,temperature,and initial concentration on the adsorption of aspirin.For this study,the carbon black obtained from the pyrolysis of tire waste was used as a precursor for low-cost adsorbents.The carbon black was treated by 1 M of hydrochloric acid solution to remove ash and sulphur content.Then,the treated carbon black was modified by impregnation with PEI in one to one weight ratio within 24 hours at 65°C and then cross linked with 1%(w/v)glutaraldehyde solution for one hour.The adsorption rate of aspirin by modified carbon black was rapid from 20 minutes to 60 minutes and reached equilibrium.Hence,the optimum contact time for this study is 60 minutes with 59.96%of aspirin removal and 29.98 mg/g adsorption capacity.The best performance for pH solution,temperature,and initial concentration was observed at pH 3(26.1 mg/g),30°C(26.9 mg/g)and 20 ppm(40.96 mg/g)respectively.

Antibacterial Activity of Polyethylenimine/Carrageenan Multilayer against Pathogenic Bacteria

The multilayer of polyethylenimine (PEI) and carrageenan (k, i, l) formed by layer-by-layer assembly was investigated for its antibacterial activity against Enterobacter cloaceae, Staphylococcus aureus and Enterococcus faecalis 29505 for potential use as coating on biomaterial surface. All the multilayers exhibited growth inhibition. PEI/Iota carrageenan multilayer was effective in inhibiting the growth of the E. cloaceae, S. aureus and E. faecalis while PEI/Lambda carrageenan was effective in inhibiting the growth of E. cloaceae. Results of the paper strip test for combined action of carrageenan and PEI showed synergism with regards to bacterial growth inhibition. The multilayers had also contact-killing effect with the test organisms. The multilayer was also characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and biomolecular interaction analysis.

The structure of colloidal polyethylenimine-silica nanocomposite microparticles

Deposition of silica from an organosilane tetraethoxysilane(TEOS)onto parent polyethylenimine(PEI)microgel particles produces a novel PEI-silica nanocomposite,which possesses greater adsorption ca-pacity for copper ions than either parent material.This study explores factors governing interactions of silica with the PEI matrix,along with structural features of resulting PEI-silica composite particles,to explain their properties and determine their application potential.The influence of initial TEOS/PEI mass ratio and the duration of silica deposition on the final silica content and distribution in the composite are studied.A comparative analysis of the structural architecture of chemically etched silica remnants,original PEI-silica composite particles and the parent PEI-microgel is carried out using X-ray photo-electron spectroscopy,small-angle X-ray scattering,and electron microscopy techniques.It is found that silica sol nanoparticles are evenly distributed throughout the PEI-microgel framework and interlinked with it via electrostatic interactions,enabling a structural model of the PEI-silica nanocomposite to be proposed.The chemical stability of resulting nanocomposite particles in parallel with the parent PEI-microgel is tested and shown to be robust for more than 100 days of storage in aqueous dispersions across a range of pH conditions,highlighting the application potential for these particles in copper capture.

Carbon dioxide capture using polyethylenimine-loaded mesoporous carbons

A high efficiency sorbent for CO2 capture was developed by loading polyethylenimine （PEI） on mesoporous carbons which possessed well-developed mesoporous structures and large pore volume. The physicochemical properties of the sorbent were characterized by N2 adsorption/desorption, scanning electron microscopy （SEM）, thermal gravimetric analysis （TG） and Fourier transform infrared spectroscopy （FT-IR） techniques followed by testing for CO2 capture. Factors that affected the sorption capacity of the sorbent were studied. The sorbent exhibited extraordinary capture capacity with CO2 concentration ranging from 5% to 80%. The optimal PEI loading was determined to be 65 wt.% with a CO2 sorption capacity of 4.82 mmol-CO2/g-sorbent in 15% CO2/N2 at 75℃, owing to low mass-transfer resistance and a high utilization ratio of the amine compound （63%）. Moisture had a promoting effect on the sorption separation of CO2. In addition, the developed sorbent could be regenerated easily at 100℃, and it exhibited excellent regenerability and stability. These results indicate that this PEI-loaded mesoporous carbon sorbent should have a good potential for CO2 capture in the future.

Enhanced Survivin siRNA Delivery Using Cationic Liposome Incorporating Fatty Acid-modified Polyethylenimine

We described a novel approach for survivin fatty acid-modified polyethylenimine. A linoleic acid siRNA cellular delivery via a cationic liposome incorporating derivative of branched polyethylenimine（PEI, Mw=25 kDa）, PEI-LA, was synthesized and incorporated into the liposome. The properties of the liposome, cytotoxicity, cellular uptake of cancer cells for survivin siRNA, survivin protein downregulation levels were investigated. PEl-modified liposome showed a lower cytotoxicity and delivered survivin siRNA into HeLa cells and A549 cells efficiently com- pared with PEI-25kDa.

Cyclodextrin/polyethylenimine-based supramolecular nanoparticles for loading and sustained release of ATP

A supramolecular nanoparticle that realized the loading and sustained release of ATP was successfully constructed from sulfato-b-cyclodextrin（SCD） and polyethylenimine（PEI）. The assembly and disassembly behaviors of supramolecular nanoparticle were investigated by means of Tyndall effect,UV–vis spectroscopy, dynamic light scattering（DLS）, zeta potential and transmission electron microscopy（TEM）. Significantly, the resulting nanoparticle was disrupted with the increasing of p H and recovered to the spherical nanoparticle as the p H decreased to initial value. Owing to the positive zeta potential, the supramolecular nanoparticle showed the good loading and sustained release abilities towards ATP.

Preparation and Characterization of Thermoresponsive Hyperbranched Polyethylenimine with Plenty of Reactive Primary Amine Groups

Certain amount of primary amine （NH2） groups of hyperbranched polyethylenimine （HPEI） was first protected by Boc groups. Subsequently, the residual reactive amine groups were reacted with isobutyric anhydride to introduce isobutyramide （IBAm） groups to HPEI. Finally, Boc groups were deprotected to result in HPEI-IBAm-NH2 with 18% of primary amine terminals on the periphery and 80% of IBAm terminal groups （abbreviated as HPEI-IBAm0.80-NH2）. 1H-NMR characterization proved the successful preparation of the product in each step. Compared with its spatial isomer HPEI- IBAm0.8o without primary amine groups, IH-NMR spectra verified that more IBAm groups were located in the interior of HPEI-IBAm0.80-NH2. The further modification of HPEI-IBAmo.so-NH2 and HPEI-IBAmo.8o with p-nitrobenzaldehyde demonstrated that HPEI-IBAm0.so-NH2 was more reactive than HPEI-IBAm0.80 due to its possession of primary amines. Turbidimetry measurements showed that HPEI-IBAm0.80-NH2 was thermoresponsive in water. In the pH range of 9.5-10 its cloud point temperature （Top） was constant, and it increased obviously upon decreasing the pH below 9.5. The thermoresponsive HPEI-IBAmo.8 exhibited the similar trend, but the pH threshold to achieve the constant Top was around 8.5. Moreover, HPEI-IBAm0.8-NH2 showed higher Top and broader phase transition than HPEI-IBAm0.8. The mechanism leading to the different thermoresponsive properties between HPEI-IBAm0.8-NH2 and its spatial isomer HPEI-IBAm0.8 was discussed.

Polyethylenimine-cyclodextrin-tegafur conjugate shows anti-cancer activity and a potential for gene delivery

Polyethylenimine-cyclodextrin-tegafur(PEI-CyD-tegafur) conjugate was synthesized as a novel multifunctional prodrug of tegafur for co-delivery of chemotherapeutic agent tegafur and enhanced green fluorescent protein(EGFP) reporter plasmid DNA.Conjugation of tegafur to PEI-CyD via chemical linkage was characterized by 1 H NMR spectrometry and ultraviolet(UV) spectrometry.PEI-CyD-tegafur was able to condense plasmid DNA into complexes of around 150 nm with positive charge at the N/P ratio of 25,in accordance with electron microscopy observation of compact and monodisperse nanoparticles.The results of in vitro experiments showed enhanced cytotoxicity and considerable transfection efficiency in B16F10 cell line.Therefore,PEI-CyD-tegafur may have great potential as a co-delivery system with anti-cancer activity and potential for gene delivery.

Effective and selective adsorption of uranyl ions by porous polyethylenimine-functionalized carboxylated chitosan/oxidized activated charcoal composite

Composite materials have elicited much interest because of their superior performance in the removal of toxic and radioactive uranyl ions from aqueous solutions.With polyethyleneimine as a functional group,carboxylated chitosan as a matrix,and oxidizing activated carbon as a nanofiller,this study synthesized a novel environment-friendly polyethylenimine-functionalized carboxylated chitosan/oxidized activated charcoal(PCO)biocomposite with a unique three-dimensional porous structure.PCO was synthesized through an easy chemical cross-linking method.Detailed characterization certified the formation of the unique three-dimensional porous structure.The obtained PCO was used to remove uranyl ions from an aqueous solution,demonstrating the maximum adsorption capacity of 450 mg·g^(−1).The adsorption capacity of PCO decreased by less than 7.51%after five adsorption-desorption cycles.PCO exhibited good adsorption selectivity(K_(d)=3.45×10^(4) mL·g^(−1))for uranyl ions.The adsorption mechanism of PCO was also discussed.The material showed good potential for application in the treatment of wastewater containing uranyl ions.

Influences of Hyperbranched Polyethylenimine on the Reactive Compatibilization of Polycarbonate/Polyamide Blends

The influences of hyperbranched polyethylenimine （hPEI）, which possesses many reactive amino end-groups, on the blending properties of bisphenol-A polycarbonate （PC） and amorphous polyamide （aPA） were systematically investigated. Scanning electron microscopy （SEM） and differential scanning calorimetry （DSC） were used to observe the effect of hPE1 on morphologies of PC and aPA phases in bulk blends. While the interfacial fracture toughness between planar PC and aPA layers with and without hPEI was studied by using augmented double cantilever beam （ADCB） method. Results show that the compatibility in PC/aPA blends can be significantly improved by adding a small amount of hPEI, mainly due to the interchange reactions between the polymers leading to the formation of block copolymers, cross-linked polymers and molecules with other constitutions. The augmented double cantilever beam experiments showed that the reactive process drastically reinforced the interfacial adhesion between planar layers of PC and aPA. However, degradation takes place during annealing at 180℃, which was responsible for the production of small molar mass species of PC.

Catalysis of Phosphate by a Modified Polyethylenimine

During the past three decades,in order to mimic the action of serineproteases manymodel enzymes have been synthesized based on micellae,imidazole and polyethyleni-mines.In this note,we describe a new protease model which can(i)noncovalently bindester substrates,(ii)accept their phosphoryl groups and(iii)dephosporylate to regeneratethe original catalytic entity.It is a true catalyst.Model 1 possesses oxime groups and long hydrocarbon chains linked to amine nitro-gen of polyethylenimine.This polymer has some attractive features:(i)Being amphiphilic,1 forms intramolecular micellae which is capable of associating with esters and othersubstrates.(ii)It has very strong binding affinity for anions and marked esters.(iii)

Influence of Aliphatic Amide Terminals on the Thermoresponsive Properties of Hyperbranched Polyethylenimines

Acetamide （C2）, propionamide （C3）, butyramide （C4）, isobutyramide （i-C4）, isovaleramide （i-C5） and trimethylacetamide （t-C5） groups each were introduced to the terminals of hyperbranched polyethylenimine （HPEI） through the amidation reaction between HPEI and the corresponding anhydride. Moreover, HPEIs terminated with two kinds of amides were also prepared. The first amide was fixed to be i-C4 with 52% degree of amidation （DA）, and the second amide varied from C2, C3, C4, i-C5 to t-C5. All the polymers were characterized by 1H-NMR. Turbidimetry measurements were performed for these polymers in water at different temperatures. With respect to the polymers bearing only one kind of amide group, except C2, all the other amide groups could render thermoresponsive properties to HPEI. The specific ordering of these amide groups to reduce the cloud point temperature （Top） was as follows： i-C5 〉 t-C5 〉 C4 〉 i-C4 〉 C3. Moreover, the more branched i-C4 and t-C5 were better groups than their less branched isomers C4 and i-C5 in the Tcp range of 12-51 ~C to render the sharper phase transition to the thermoresponsive polymers. As for the polymers bearing two kinds of amide groups, the further introduction of C2, C3, C4, i-C5 or t-C5 could effectively endow HPEI bearing 52% of i-C4 with thermoresponsive properties. The specific ordering of these second amide groups to reduce the Top was as follows： i-C5 〉 C4 〉 i-C4 〉 C3 〉 C2. C4, i-C5 and t-C5 were all effective second amide groups to prepare the thermoresponsive polymers with sharper phase transition.

Tetraphenylethene End-capped Polyethylenimine Fluorescent Nanoparticles for Cell Imaging

Water soluble tetraphenylethene-based（TPE） aggregation-induced emission fluorescent organic nanoparticles（FONs） were facilely prepared via Schiff base condensation with polyethylenimine（PEI） and subsequent reduction. The obtained TPE-PEI FONs were characterized by a series of techniques including 1H-NMR, 13C-NMR, gel permeation chromatography, UV absorption spectra, fluorescence spectra, Fourier transform infrared spectroscopy, size distribution and zeta potential measurement, and transmission electron microscopy. Biocompatibility evaluation and cell imaging of TPE-PEI FONs were further explored. We demonstrated that such FONs showed intense fluorescence, spherical morphology and excellent biocompatibility, making them very suitable for cell imaging application.

SYSTHESIS OF DOUBLE-HYDROPHILIC CORE-SHELL TYPE MULTIARM STAR COPOLYMER POLYETHYLENIMINE-block-POLY(2-HYDROXYETHYL METHACRYLATE)

Multiarm star block copolymers hyperbranched polyethylenimine-b-poly（2-hydroxyethyl methacrylate） （HPEI-b- PHEMA） with average 28 PHEMA arms have been prepared by atom transfer radical polymerization （ATRP） of HEMA in a mixed solvent of methanol and water using a core-first strategy. The hyperbranched macroinitiator employed was prepared on the basis of well-defined hyperbranched polyethylenimine with Mw/Mn of 1.04 by amidation with 2-bromo-isobutyryl bromide. The polymerization condition was optimized to prepare star copolymers with narrow dispersity, and the variables included the volume ratio of methanol to water, the molar ratio of initiating site to CuC1 and the molar ratio of [CuCl]：[CuBr2]. Under the optimized polymerization condition, the lowest Mw/Mn value of the obtained star copolymers was around 1.3. Kinetic analysis showed that an induction period existed in the polymerization of HEMA. After this induction period, a linear dependence of ln（[M]0/[M]t） on time was observed. The obtained HPEI-b-PHEMA could adsorb hydrophilic molecules. The comparison with the star copolymer with hydrophobic core and hydrophilic PHEMA shell verified that both the hydrophilic core and shell could host the hydrophilic guests, but the amidated HPEI core was more effective than the PHEMA shell.