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Yang C.,Lanzhou Military Command Center for Disease Control and Prevention | Peng R.,Soochow University of China
Macromolecular Research

1.8 kDa branched polyethylenimine (PEI) was modified with pH-sensitive degradable acetal containing hydrophobe, 2,4,6-trimethoxybenzylidene- tris(hydroxymethyl) ethane (TMB-THME), to enhance its DNA condensation under extracellular conditions as well as to achieve active DNA release inside cells. PEI-(TMBTHME) n conjugates in the amount of 1.8 kDa were prepared with varying degrees of substitution (DS) from 3.0, 5.7 to 10.1. Notably, dynamic light scattering (DLS) measurements showed that all three 1.8 kDa PEI-(TMB-THME) n conjugates could effectively condense DNA into nano-sized particles (189-197 nm) at N/P ratios ranging from 20/1 to 80/1. The surface charges of PEI-(TMB-THME) n polyplexes depending on DS and N/P ratios varied from +22 to +28 m V, which were comparable to or slightly higher than the unmodified 1.8 kDa PEI counterparts (∼+22 to +23 mV). Under a mildly acidic condition mimicking that of endosomes, interestingly, 1.8 kDa PEI-(TMB-THME) n polyplexes were quickly unpacked to release DNA because of the pH-induced acetal degradation that transforms hydrophobic modification into hydrophilic modification. MTT assays demonstrated that all PEI-(TMB-THME) n polyplexes displayed low cytotoxicity (>80%) to 293T, and HeLa cells at N/P ratios ranging from 20/1 to 60/1. The in vitro gene transfection studies showed that the transfection activity of 1.8 kDa PEI was significantly enhanced by modifications with TMB-THME, in which transfection efficiencies increased with increasing DS. For example, 1.8 kDa PEI-(TMB-THME) 10.1 polyplexes displayed 250-fold and 80-fold higher transfection efficiencies than those of the unmodified 1.8 kDa PEI counterparts in 293T and HeLa cells, respectively, which were approximately 4-fold and 2-fold higher than that of 25 kDa PEI control. The superior transfection activity of 1.8 kDa PEI-(TMB-THME) 10.1 polyplexes was also confirmed by confocal laser scanning microscopy (CLSM), which showed efficient delivery of DNA into the nuclei of 293T cells following 4 h transfection. Modification of low molecular weight PEI with pH-sensitive degrad-able hydrophobe has appeared to be highly promising in the development of "artificial viruses" for safe and efficient gene transfer. Source

Zhong Z.,Soochow University of China | Zheng M.,Soochow University of China | Zhong Z.,Lanzhou Military Command Center for Disease Control and Prevention | Zhou L.,Soochow University of China | And 2 more authors.

Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ∼ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications. © 2012 American Chemical Society. Source

Luo X.-H.,Lanzhou Military Command Center for Disease Control and Prevention | He Z.-P.,Lanzhou Military Command Center for Disease Control and Prevention
Chinese Journal of Endemiology

Objective: In order to offer scientific evidence for prevention and therapy of Lyme disease, we had investigated the natural infection of lyme disease Borrelia burgdorferi(Bb) of 4 ticks in Diebu and Huajian areas of Gansu province. Methods: Epidemiology detection of natural infection of lyme disease Bb was accomplished for four dominant tick(Dermacetor silvarum, Dermacentor nuttalli, Haemaphysalis Japonica and Ixodes Crenulatus) at Diebu, Diebu forest zone(Qin mountain), north of Min Mountains of Huajian region and Huajian forest zone, north of Qilian Mountains of Sunan region, Gansu, from March to June, 2010. The methods of dispersion of clamp every 10 m which was put in the morning and retrieved at night were used to capture rodent animal and gnawer retrorse hair inspection insect methods were used to collect parasitic tick. Flagging methods were used to collect free tick. Four kinds of live adult ticks were dissected after cleaning and disinfection. Intestinal contents were smeared, lyme disease Bb was observed under dark-field microscope. Etiological agent was cultivated and separated. Separated spirochete was confirmed by spirochete monoclonal and polyclonal antibody. Results: The ticks collected were classified as 2 families, 8 genera, 36 species, i.e. Ixodidae 6 genera, 33 species and Argasidae 2 genera, 3 species. The method of dark-field microscope was used to detect the lyme disease spirochete in 201 ticks intestines after dissection of Dermacentor silvarum, Dermacentor nuttalli, Haemaphysalis Japonica and Ixodes Crenulatus, the lyme disease Bb-positive were 25 with positive rate of 12.44% (25/201). The lyme disease Bb was cultivated and separated from 12 ticks with the positive rate of 18.46%(12/65) after inoculation and cultivation of 3 species, 65 ticks of Dermacentor silvarum, Dermacentor nuttalli and Haemaphysalis Japonica. Conclusions: Dermacentor silvarum, Dermacentor nuttalli and Haemaphysalis Japonica have different degrees of natural infection of lyme disease Bb. Source

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