Liposome cationic

Reports of CNS gene transfer using cationic liposomes began to appear in the literature from the early 1990s. DNA-liposome complexes (lipoplexes) 

Cationic liposomes have also been used for treating neurodegenerative disease in in vivo models of Parkinson s disease and EAE. For example, EAE symptoms were inhibited by a single injection of therapeutic cytokine (IL-4, IFNp and TGF(3) lipoplexes directly into the CNS. Table 17.3 (below) summarizes gene delivery vectors used in MS. A list of cationic lipid/liposomes enabling in vivo and ex vivo transfection to the CNS is provided in Table 17.2. 

Cationic polymers also exhibit DNA condensing properties similar to cationic lipids. These polymers can be divided into two categories based on their origin natural and synthetic. 

A list of natural cationic polymers enabling in vivoHn vitro gene therapy to the brain is provided in Table 17.3. 

Gene delivery vector Animal species In vivo Ex vivo Time points Dose Target region Ref. 

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Borgatti M., Breda L., Cortesi R., Nastruzzi C., Romanelli A., Saviano M., Bianchi N., Mischiatic C, Gambari R. Cationic liposomes as delivery systems for double-stranded PNA-DNA chimeras exhibiting decoy activity against NF-kB transcription factors. Biochem. Pharmacol. 2002 64 609-616. 

Cationic liposomes form complexes with plasmid DNA. Also, in this case DOPE affects the architecture of the complexes. DOTAP liposomes without DOPE are able to condense DNA, while DOTAP/DOPE liposomes do not condense DNA, presumably due to Hn-phase formation [230,233]. Interestingly, multivalent cationic liposomes, such as DOGS, can condense DNA more efficiently than monovalent cationic liposomes [233]. If the complexation is carried out at high ionic strength the DNA condensation is less prominent than in plain water. 

In this respect, an interesting approach to reduce degradation and possible toxicity problems related to nucleic acid use in vivo is offered by their encapsulation in or association to microcarrier systems, such as neutral or cationic liposome and polymeric microparticles [41 14],... 

Wasan, E.K., Fairchild, A., and Bally, M.B., Cationic liposome-plasmid DNA complexes used for gene transfer retain a significant trapped volume, Journal of Pharmaceutical Science, 1998, 87, 9-14. 

Miller, A.D., The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy, Current Medicinal Chemistry, 2003, 10, 1195-1211. 

Sternberg, B., Sorgi, F.L., and Huang, L., New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy, FEBS Letters, 1994, 356, 361-366. 

Budker V, Gurevich V, Hagstrom JE, Bortzov F, Wolff JA (1996) pH-sensitive, cationic liposomes a new synthetic virus-like vector. Nat Biotechnol 14 760-764... 

One problem with the ATP assay in aqueous media is that the enzyme requires hydrophobic media the reaction rate of luciferase-catalyzed reactions is variously affected by the presence of detergents [117, 118]. The presence of cationic liposomes improves sensitivity by a factor of five times compared to that in water alone [119]. 

Formation of a complex between DNA and polycationic compounds appears to be the initial and quite possibly a critical parameter for nonviral gene delivery. Several synthetic vector systems, which are generally cationic in nature, including poly(lysines), cationic liposomes or various types of block copolymers and recently dendrimers, have been shown to self-assemble with plasmid DNA [13-15] [16]. Specific physicochemical properties manifested by these DNA complexes depend on the type of cationic agent used however, interesting patterns for such interactions are beginning to evolve [17, 18]. Under certain conditions, the interaction of DNA with polyvalent cations results in... 

Duncan JE, Whitsett JA, Horowitz AD (1997) Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro. Hum Gene Ther 8(4) 431-438... 

Figure 7 Pharmacokinetic properties and in vivo gene expression of stabilized plasmid-lipid particles (SPLP). (A) The levels of intact plasmid DNA (pDNA) in the circulation resulting from IV injection of naked plamid pDNA ( ), lipoplexes (O), and SPLP ( ) were determined by Southern blot analysis of plasma samples (100 pg pDNA/mouse). (B) Transgene expression at a distal tumor site resulting from rv injection of naked plamid pDNA ( ), plamid pDNA-cationic liposome complexes (O), and SPLP ( ).

Gao X, Huang L. Cationic liposome-mediated gene transfer. Gene Ther 1995 2 710. 

Maurer N, Wong KF, Stark H, et al. Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol destabilized cationic liposomes formation of small multilamellar liposomes. Biophys J 2001 80 2310. 

Up to 500 pg of plasmid DNA (for the amount of PC shown above) is dissolved in 2mL distilled water, or lOmM sodium phosphate buffer (PB) of pH 7.2 if needed. For liposomes containing both the plasmid DNA and the vaccine protein it encodes (or only the protein), up to 1 mg of the protein is included. The nature of buffer with respect to composition, pH, and molarity can be varied as long as this does not interfere with liposome formation or DNA and protein entrapment yield. Amounts of added DNA and protein can be increased proportionally to the total amount of lipid used. For cationic liposomes, the amount of added DNA can also be increased by employing more cationic lipid. 

S-labeled plasmid DNA (10-500 pg) was incorporated into( or mixed ( ) with neutral (PC, DOPE), anionic (PC, DOPE, PS, or PG), or cationic (PC, DOPE, SA, BisHOP, DOTMA, DC-CHOL, DOTAP, or DODAP) DRV. Incorporation values of the different amounts of DNA used for each of the liposomal formulations did not differ significantly and were therefore pooled (values shown are means of values obtained from three to five experiments). PC (16pmol) was used in molar ratios of 1 0.5 (neutral) and 1 0.5 0.25 (anionic and cationic liposomes). 

The content of vaccine within the small liposomes is estimated as in the section Estimation of Vaccine Entrapment in Dehydration-Rehydration Vesicles Liposomes for both microfluidized and sucrose liposomes and expressed as percentage of DNA and/or protein in the mixture subjected to freeze drying as in the section Preparation of Vaccine-Containing Small Liposomes by the Sucrose Method in the case of sucrose small liposomes or in the original DRV preparation (obtained in the section Estimation of Vaccine Entrapment in DRV Liposomes ) for microfluidized liposomes. Vesicle size measurements are carried out by PCS as described elsewhere (6,8,17). Liposomes can also be subjected to microelectrophoresis in a Zetasizer to determine their zeta potential. This is often required to determine the net surface charge of DNA-containing cationic liposomes. 

It has been demonstrated that the unmethylated CpG motifs in the DNA activate DCs through TLR9, but association with the cationic liposome is required for full activation of these cells. Because the liposomes alone do not generate a cytokine response, we can suppose there is a second receptor... 

Miller AD. Cationic liposomes for gene therapy. Angew Chem Int Ed Engl 1998 37 1768. 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

For automation, the lipofection process was split of into four independent parts as follows (i) preparation of cationic liposomes, (ii) formation of lipo-plexes, (iii) transfection of the cells, and (iv) quantification of the lipofection efficiency and lipofection-induced cytotoxicity. As shown in Figure 1, this subdivision corresponds to the typical lipofection procedure and each part can be performed separately. 

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