γ-Ray-Radiation-Scissioned Chitosan as a Gene Carrier and Its Improved in uitro Gene Transfection Performance

Chitosan (CS) is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecular weight (MW) of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can e ectively bind with plasmid (pEGFP) through complex coacervation method, forming pEGFP/ γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in uitro gene transfection efficiencies of the pEGFP/ γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.     

重离子辐射提高腺病毒介导小鼠黑色素瘤细胞转染率的研究

探讨12C6+ 离子束辐射对用带有绿色荧光蛋白基因的缺陷性腺病毒（AdCMV GFP）转染小鼠黑色素瘤细胞(B16细胞系)的影响。 采用不同剂量的12C6+ 重离子束辐射经AdCMV GFP 转染的B16细胞， 利用流式细胞仪检测腺病毒的转染率。 结果表明， 12C6＋重离子束辐射能提高腺病毒对B16细胞的转染率， 且具有量效关系。 此外， 先转染后辐射法比起先辐射后转染法能更显著地提高转染率。The effect of 12C6+ beam irradiation on AdCMV GFP (a replication deficient recombinant adenoviral vector containing CMV promoter and green fluorescent protein) gene transfection efficiency for murine melanoma cell B16 has been investigated. B16 cells infected with AdCMV GFP were irradiated by different doses of 12C6+ beam. The transfection efficiency was assessed by flow cytometry (FCM). Results show that 12C6+ beam irradiation can improve tansfection efficiency of AdCMV GFP on murine melanoma cell B16 in a dose dependent manner. In addition， the tansfection efficiency in pre tranfection plus irradiation group is higher than that in pre irradiation plus tranfection group at the same dose irradiation dose.     

A Novel Approach for Introducing Bio-Materials Into Cells

A novel approach was developed to introduce biological materials into cells for gene transfection and gene therapy applications. The method is based on the technique of electrospraying bio-materials into cells. A prototype apparatus was constructed for a feasibility study. The features of the gene transfector include: (1) A dual-capillary assembly to spray suspensions of biological materials. The outer capillary provided sheathing liquid that controlled the charge level on individual particles without altering the properties of suspensions. (2) An air–CO₂ gas mixture was used for suppressing possible corona discharge and kept the same gas composition as those in incubators. (3) The designed chamber enabled the spray to operate at reduced pressure for increasing sprayed particle velocity. In the feasibility study, both suspensions of plasmid and plasmid-coated gold particles were used. The plasmid used was the commercially available Enhanced Green Fluorescent Protein gene. COS-1 cells were used as the target and the liquid media was evacuated immediately prior to the spraying process. Electrospraying was conducted at ambient pressure and the duration was no more than 2 min. After the spray transfection, the media was immediately replaced and the cell samples were returned to the incubator for 36 h. Transgene expression was detected by cellular fluorescence. This technology promises to have great potential for gene transfection and therapy studies.     

Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non‐viral Oligonucleotide Delivery Carriers

A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single‐stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single‐stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast‐like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron‐functionalised material without affecting the cellular viability.     

Hydrophobic polyphenylalanine-grafted hyperbranched polyethylenimine and its in vitro gene transfection

A series of amphiphilic multi‐armed PPn copolymers were prepared by ROP of Phe‐NCA with PEI‐25k as a macroinitiator. The particle size of the PPn/DNA complexes was about 100 nm and the zeta potentials were below 20 mV. An MTT assay demonstrated that all the PPn copolymers had lower cytotoxicity compared to PEI‐25k. In vitro gene transfection studies were also conducted in HeLa, 293 and CT 26 cells. The optimal quantity of hydrophobic phenylalanine segments in PP80 led to higher transfection efficiency in various cell lines based on this study. The results indicate that PP80 was the best candidate for gene delivery among these PPn copolymers.

     

Superhydrophobic-Superhydrophilic Micropatterning: Towards Genome-on-a-Chip Cell Microarrays

High-density cell microarrays based on superhydrophilic microspots separated by superhydrophobic barriers have been realized. The microspots absorb water solutions, while the barriers prevent cross-contamination, thus allowing the spots to be used as reservoirs for transfection mixtures and preventing cell proliferation and cell migration between the microspots. The picture shows four cell types after two days of culturing on the microarray.     

Structure–Function Relationships of New Lipids Designed for DNA Transfection

Cationic liposome/DNA complexes can be used as nonviral vectors for direct delivery of DNA‐based biopharmaceuticals to damaged cells and tissues. To obtain more effective and safer liposome‐based gene transfection systems, two cationic lipids with identical head groups but different chain structures are investigated with respect to their in vitro gene‐transfer activity, their cell‐damaging characteristics, and their physicochemical properties. The gene‐transfer activities of the two lipids are very different. Differential scanning calorimetry and synchrotron small‐ and wide‐angle X‐ray scattering give valuable structural insight. A subgel‐like structure with high packing density and high phase‐transition temperature from gel to liquid‐crystalline state are found for lipid 7 (N′‐2‐[(2,6‐diamino‐1‐oxohexyl)amino]ethyl‐2,N‐bis(hexadecyl)propanediamide) containing two saturated chains. Additionally, an ordered head‐group lattice based on formation of a hydrogen‐bond network is present. In contrast, lipid 8 (N′‐2‐[(2,6‐diamino‐1‐oxohexyl)amino]ethyl‐2‐hexadecyl‐N‐[(9Z)‐octadec‐9‐enyl]propanediamide) with one unsaturated and one saturated chain shows a lower phase‐transition temperature and a reduced packing density. These properties enhance incorporation of the helper lipid cholesterol needed for gene transfection. Both lipids, either pure or in mixtures with cholesterol, form lamellar phases, which are preserved after addition of DNA. However, the system separates into phases containing DNA and phases without DNA. On increasing the temperature, DNA is released and only a lipid phase without intercalated DNA strands is observed. The conversion temperatures are very different in the two systems studied. The important parameter seems to be the charge density of the lipid membranes, which is a result of different solubility of cholesterol in the two lipid membranes. Therefore, different binding affinities of the DNA to the lipid mixtures are achieved.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

Synthesis, characterization, and remarkable biological properties of cyclodextrins bearing guanidinoalkylamino and aminoalkylamino groups on their primary side

The introduction of aminoalkylamino and guanidinoalkylamino substituents on the primary side of beta- and gamma-cyclodextrin (CDs) resulted in a series of novel compounds that were extensively characterized by NMR spectroscopy and mass spectrometry. Bromination of the primary side of beta- and gamma-CD, and reaction with neat alkylene diamines at a pressure of 7 atm afforded aminoalkylamino derivatives that were then guanylated at the primary amino group to give the corresponding guanidinoalkylamino-CDs. These compounds are water soluble and display pK(a) values that allow them to be mostly protonated at neutral pH; for example, pK(a(1)) approximately 6.4 and pK(a(2)) approximately 9.5 for the aminoethylamino-beta-CD and pK(a(1)) approximately 7.8 and pK(a(2)) approximately 11.0 for the guanidinoethylamino-beta-CD. The title CDs are rigid, cyclic alpha-D-glucopyranose oligomers (heptamers or octamers) with branches that resemble lysine and arginine side chains that enable multiple interactions with suitable substrates. Thus, they bear similarities to known cell-penetrating peptides. Indeed, the compounds were found to cross the membranes of HeLa cells and penetrate inside the cytoplasm quickly, the guadinylated ones within 15 min, as shown by fluorescence microscopy using fluorescein-labeled derivatives. The toxicity of the compounds, measured by performing MTT tests, ranged from 50 to 300 microM. Furthermore, some of the aminated CDs could facilitate the transfection of DNA expressing the green fluorescent protein (GFP) in HEK 293T cells, with effectiveness comparable to the commercial agent Lipofectamine 2000. Circular dichroism, atomic force microscopy and electrophoresis experiments confirmed the strong interaction of the compounds with DNA. Because of their carbohydrate, non-peptide nature the title compounds are not anticipated to be enzymatically labile or immunogenic, and thus they fulfill many of the criteria for non-hazardous transport vectors in biological and pharmaceutical applications.