Kyonghee Son

Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged

In medium where in vitro transfection is routinely performed, DC-chol liposomes alone were nearly neutral, whereas the DC-chol liposome/DNA complexes were largely negatively charged which changed only slightly at all [liposome]/[DNA] ratios (zeta=-27.1 to -21.8 mV). Three other commercial transfection reagents, Lipofectin(R), LipofectAMINE 2000, and SuperFect, were also largely negatively charged when complexed with DNA. The aggregation of liposomes in medium was prevented by the addition of DNA. Incubation of the complexes in medium did not change their size, charge or lipofection activity for 30 min. These results suggest that, in medium, the liposome/DNA complexes were formed at the time of mixing with negative charges.

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.

A nitric oxide production bioassay for interferon-γ

Interferon-gamma (IFN-gamma), produced by stimulated T lymphocytes and natural killer cells, has an antitumor and antiviral activity by inhibition of cell growth, immunomodulation, and/or activation of macrophages. Although several IFN-gamma assays have been used, there are no simple, inexpensive, and specific assays. We have developed a new bioassay for IFN-gamma which measures the concentration of nitric oxide (NO) generated by a macrophage cell line RAW264.7 stimulated with IFN-gamma. NO production, determined by nitrite (NO2-) accumulation in culture medium, was linear at IFN-gamma concentrations between 0 and 10 U/ml and logarithmically linear between 2 and 100 U/ml, when RAW cells (1 x 10(5) cells/200 microliters/well in 96 well plate) were incubated with murine recombinant IFN-gamma for 24 h. The new assay has a high sensitivity with the detection limit of 0.1-0.2 ng/ml IFN-gamma, which is similar to that of the enzyme-linked immunosorbent assay (ELISA) and antiviral assays. Other cytokines such as IFN-alpha, IFN-beta, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-2, and IL-6, either alone or in combination did not produce NO from RAW264.7. Exogenous NO2-, NO3- or NO2- plus NO3- did not interfere with the IFN-gamma-induced NO formation as well as NO2- measurement. The IFN-gamma bioactivity, measured by the RAW264.7 bioassay, expressed from the transfected human ovarian tumor cells was closely correlated with the levels of IFN-gamma protein concentration measured by the ELISA. Therefore, our new method can be applicable for monitoring IFN-gamma gene expression and accumulation in culture medium for IFN-gamma therapy.

Nitric oxide-mediated tumor cell killing of cisplatin-based interferon-γ gene therapy in murine ovarian carcinoma

We have determined the role of nitric oxide (NO)-mediated tumor cell killing in the treatment of an animal model of murine ovarian carcinoma grown in the peritoneum with a combination of cisplatin and cationic liposomes containing an expression vector for interferon-γ (IFN-γ). The approach was to determine whether the therapy was effective in mice homozygous for a disrupted inducible NO synthase (iNOS) allele; these mice were unable to produce NO in response to IFN-γ. iNOS (−/−) mice treated with both cisplatin and liposomal IFN-γ gene did not produce a significant amount of NO in ascites (12.1 ± 4.5 μM), although they expressed a high level of IFN-γ (9002 ± 723 U/mL of ascitic fluid). As a result, mice died of tumors within 11–62 days. However, wild-type mice treated with both cisplatin and liposomal IFN-γ gene produced a significant amount of NO in ascites (113.7 ± 17.9 μM) with a high level of IFN-γ gene expression (9350 ± 1254 U/mL of ascitic fluid) and were free of tumors for at least 80 days. This result confirmed that NO was a direct mediator of IFN-γ cytotoxicity.

Delipidated serum abolishes the inhibitory effect of serum on in vitro liposome-mediated transfection

All the standard in vitro lipofection has been routinely performed in serum-free medium as the transfection activity of liposome/DNA complexes is sensitive to the presence of serum. In this study, we have demonstrated that lipid-rich serum lipoprotein included in the transfection medium strongly inhibited the transfection activity of DC-chol liposome/DNA complexes in five different cell types (CHO, 293, A2780CP, A431 and SKBR3). The levels of inhibition by serum lipoprotein were rather greater than those by serum and varied with cell types. However, this inhibition was completely abolished by delipidation of serum. Thus, delipidated serum can be included in the transfection medium. The complexes formed in the presence of serum (zeta=-18.2+/-1.07 mV), delipidated serum (zeta=-19.6+/-0.54 mV), IgG (zeta=-21.6+/-1.92 mV) or serum lipoprotein (zeta=-10.5+/-2.33 mV) were as much negatively charged as those in serum-free medium (zeta=-21.3+/-1.60 mV). The results suggest that the inhibition of liposome-mediated transfection by serum was not associated with charges of serum proteins but with lipids or lipid-associated proteins present in serum.

Chemical Toxicants Activate Murine Ovarian Ascitic Tumor Cells for In Situ Lipofection

Cisplatin-induced enhancement of lipofection was tested for the involvement of cell death via reactive oxygen species (ROS).A direct injection of exogenous ROS into tumor-bearing mice significantly sensitized tumor cells for in situ lipofection. Chemotherapeutic drugs, independent of ROS production, also significantly facilitated transduction, which was maximum at nontoxic or subtoxic doses and was host independent. Transplatin and pro-oxidants, having no antitumor activity, significantly facilitated transduction, while antioxidants had little effect. These results suggest that cisplatinfacilitated lipofection in tumor cells may be a general phenomenon of toxic chemicals inducing cell death in a mammalian system.

Overexpression of protein kinase A – RIα reduces lipofection efficiency of cisplatin-resistant human tumor cells

Cisplatin-resistant variant A2780CP/vector cells were 4.0-5.3-fold more transfectable and 7.6-fold more resistant to cisplatin than their parent cisplatin-sensitive human ovarian carcinoma A2780/vector cells. Overexpression of cAMP-dependent protein kinase Type I regulatory alpha subunit (PKA-RIalpha) gene in A2780CP cells significantly reduced (maximum 47.0%) the transfection activity, with a slight reduction (maximum 27.3%) of cisplatin resistance, of A2780CP cells. However, RIalpha-overexpressing A2780CP (A2780CP/RIalpha) cells were still 2.5-to 3.0-fold more transfectable and 5.5-fold more resistant to cisplatin than A2780 cells. This results suggest that gene transfer efficiency is associated with cisplatin resistance, in part, through the PKA-mediated cAMP signal transduction pathway.