On the disulfide-linker strategy for designing efficacious cationic transfection lipids: an unexpected transfection profile

AbstractHerein, employing a previously reported disulfide-linker strategy, we have designed and synthesized a novel cationic lipid 2 with a disulfide-linker and its non-disulfide control analog lipid 1. The relative efficacies of lipids 1 and 2 in transfecting CHO, COS-1 and MCF-7 cells were measured using both reporter gene and whole cell histochemical staining assays. In stark contrast to the expectation based on the disulfide-linker strategy, the control non-disulfide cationic lipid 1 showed phenomenally superior in vitro transfection efficacies to its essentially transfection incompetent disulfide counterpart lipid 2. Results in DNase I protection experiments and the electrophoretic gel patterns in the presence of glutathione, taken together, are consistent with the notion that the success of the disulfide-linker strategy may depend more critically on the DNase I sensitivity of the lipoplexes than on the efficient DNA release induced by intracellular glutathione pool

Tumor detection and elimination by a targeted gallium corrole

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents

New histidylated cationic lipids for DNA and mRNA- based lipofection

Plasmid delivery into the cytosol remains one of the limiting factor to achieve efficient transfection. We have previously demonstrated that the presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers significantly enhances their gene delivery efficiencies. We designed and synthesized two novel non-glycerol and a cholesterol based histidylated cationic amphiphiles containing a single histidine head group. Physico-chemical characteristics of all the novel liposomes and lipoplexes including lipid:DNA interactions, global surface charge, sizes, etc. were evaluated. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-Histidine(N,N-di-n-hexadecylamine, N-methyl)ethylamide (lipid 2) in combination with cholesterol and Cholesteryl-L-Histidine-Ethylamide (lipid 3) in combinaison with DOPE gave efficient DNA and mRNA transfections into various cell lines. DNA transfection efficiency into A549, 293T7 and HeLa cells of Chol/lipid 1 lipoplexes was similar with that of FuGENE6 and DC-Chol lipoplexes but was two order of magnitude higher in HepG2 cells. Membrane fusion activity measurements using FRET technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. By using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter, we showed that the release of DNA from the endosomally trapped lipoplexes to the cytosol is acidic dependent and presumably mediated by the imidazole ring protonation of histidine head group of these cationic amphiphiles. A better efficiency was obtained with Chol/lipid 2 lipoplexes than with Chol/lipid 1 lipoplexes when using the cytosolic luciferase expression vector. As anticipated, transfection efficiency of Lipid 3 was greatly inhibited in the presence of Bafilomycin A1. By contrast, endosome escape of DNA with a new cholesterol based cationic lipid containing no histidine head-group (Alanine-Cholesteryl-Ethylamide; lipid 4) seemed to be independent of endosome acidification. However, transfection efficacy of lipids 3 & 4 was similar. In conclusion, we show that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds or cholesterol is sufficient to impart remarkable transfection properties on the resulting cationic amphiphile via endosome-disrupting characteristics of the histidine functionalities Figure 1

Histidylated lipid-modified sendai viral envelopes mediate enhanced membrane fusion and potentiate targeted gene delivery

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p <0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals