Liposome cationic preparation

The photophysical properties of magnesium(II) tetra-(i-butyl)phthalocyanine (27) have been studied in solution, in micelles and in liposomes cation radical formation (CBr4 as electron acceptor) has been detected with UV excitation, or by a two-photon excitation using a pulsed laser in the therapeutic window at 670 nm.118 The Mg11 complex of octa(tri-z -propylsilylethy-nyl)tetra[6,7]quinoxalinoporphyrazine (28) has been prepared as a potential PDT sensitizer. The synthesis is shown in Figure 8. Compound (28) has Amax 770 nm (e = 512,000 M-1 cm-1), d>f = 0.46 and d>A = 0.19 (all in THF, under air).119... 

Quantitative entrapment of vaccines into small (up to about 200 nm diameter) liposomes in the absence of microfluidization (which can damage DNA and other labile materials when extensive) can be carried out by a novel one-step method (7) as follows SUVs (e.g., cationic) prepared as in section Preparation of Small Unilamellar Vesicles are mixed with sucrose to give a range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appropriate amount of plasmid DNA (e.g., 10-500 pg) and/or protein (e.g., up to 1 mg). The mixture is then rapidly frozen and subjected to dehydration by freeze-drying, followed by rehydration as in section Preparation of Vaccine-Containing Dehydration-Rehydration Vesicles. ... 

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).

The therapeutic efficacy of either systemic or local pulmonary delivery of the IFN-y gene was evaluated in a murine allergen-induced airway hyperresponsiveness (AHR) model (Dow et al. 1999) and it was found that a high efficiency of gene transfer could be achieved. Intratracheal administered cationic liposomes were prepared from a mixture of l,2-diacylglycero-3-ethylphosphocholine (EDMPC) and cholesterol. Intravenous injections were prepared from l,2-dioleyl-3-trimethylammo-ninm propane (DOTAP) and cholesterol and compared with pulmonary administered... 

Liposome-mediated gene delivery is dependent on numerous factors, such as, the formulation of the liposomes including the cationic lipid/neutral lipid ratio, how the liposomes are prepared, the cationic liposome/DNA charge ratio of the complex of cationic liposome and DNA (lipoplex), and the method used to produce the lipoplex. Recently, it was reported that the way in which a liposome was prepared affected transfection efficiency (1), and formation method of lipoplex affected size of lipoplex in which large ones increased the efficiency of transfection (2-7). 

The anionic liposomes were prepared by the film method as described for the cationic liposome in Subheading 3.1. 

For preparation of lipoplexes, first of all, concentrated stocks of nncleic acid and cationic liposome are prepared and stored at optimal condition (-80°C to 4°C). Then, the concentrated stocks are diluted with required volume of diluent according to desired N/P ratio. An example calculation of N/P ratio is presented. Then, the diluted nucleic acids and liposomes are mixed and stand at room temperature for desired time to allow the lipoplex formation. The formation and stability of complexes can be assessed with the gel retardation assay. 

To use liposomes as delivery systems, drug is added during the formation process. Flydrophilic compounds usually reside in the aqueous portion of the vesicle, whereas hydrophobic species tend to remain in the lipid proteins. The physical characteristics and stability of lipsomal preparations depend on pH, ionic strength, the presence of divalent cations, and the nature of the phospholipids and additives used [45 47],... 

In the early 1960s Bangham prepared liposomes—sealed vesicles made from lecithin or other lipids, with an aqueous interior. Liposomes with purified Na/K-ATPase inserted into their membranes pumped cations in the presence of ATP, convincingly confirming the function of the ATPase, and the existence of vectorially (asymmetrically) organized proteins. 

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. 

Initial vaccination studies with LPDI nanoparticles were completed using liposomes prepared with both 1,2-dioleyltriammonium propane (DOTAP) and cholesterol. After it was determined that cholesterol played only a small structural role and was not necessary for activity, the liposomes were then prepared using only DOTAP to become an LPDI type of formulation. Regardless of the lipid used, the ratio of cationic lipid, polycation, and DNA must be maintained to have all properties associated with LPDI particles (2). 

Cationic lipids cannot be dissolved in water and form aggregates in aqueous solution, such as bilayers. To prepare a homogeneous reagent, in most cases liposomes were made from cationic lipids in a first step. When it is not possible to form stable lipid bilayers (i.e., liposomes) using a single lipid, then it may be necessary to combine the cationic lipid with one or more so-called helper lipids like cholesterol (Choi) (41) or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (42). 

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. 

For the design of mitochondriotropic liposomes, we have used a method, that has been a standard procedure in liposome technology for over 30 years the lipid-mediated anchoring of artificially hydrophobized water-soluble molecules into liposomal membranes (25-28). We have hydrophobized mitochondriotropic TPP cations by conjugating them to long alkyl residues specifically, we have synthesized stearyl TPP (STPP) salts (29). Following liposome preparation in the presence of STPP, the liposomal surface became covalently modified with TPP cations, thereby rendering these liposomes mitochondriotropic as verified in vitro by fluorescence microscopy (30). 

Nonviral vector systems are usually either composed of a plasmid based expression cassette alone ( naked DNA), or are prepared with a synthetic amphipathic DNA-complexing agent (84, 88). Gene delivery systems based on nonviral vectors mainly comprise cationic liposomes, DNA-polymer-protein complexes, and mechanic administration of naked DNA. An idealized/optimized multifunctional nonviral gene delivery system is depicted in Figure 13.4. 

Although most of the studies using nonviral vectors are being performed in animal models, O Malley and colleagues have performed IL-2 gene therapy by using nonviral vector in patients with advanced head and neck cancer. In their phase I clinical trial, cationic liposomes prepared with CMV-IL-2 plasmid were injected into the tumor. All patients completed the study but most died due to the progression of the disease. One patient exhibited a decrease in the burden of the tumor. 

We examined the biodistribution of cationic liposomes/pDNA complex following intravenous injection in mice and pharmacokinetically analyzed the data based on the clearance concept (Mahato etal., 1995a, 1997). These analyses showed that the pharmacokinetics of 32P-pDNA complexes depend on their mixing (charge) ratio, the type of cationic and helper lipids (Mahato et al., 1998). When analyzed using radioactivity counting following the injection of the complex prepared with 32P-pDNA, the tissue uptake clearance per g... 

Compared to viral vectors, the potential advantages of synthetic carriers (also called non-viral vectors) are apparent. Being synthetic, they could be made safe, non-immunogenic, easy to prepare and cost-effective. DNA delivered by these carriers may not be able to replicate or recombine into infectious forms. Among many reported non-viral carriers, including cationic polymers (Behr et al., 1989 Kukowska-Latallo et al., 1996 Wu and Wu, 1988) and cationic lipids (Feigner, 1990 Lee and Huang, 1997), the most frequently used form is cationic liposomes. 

The helper effects of DOPE and cholesterol appear to be hydrocarbon chain-specific. This is demonstrated in studies of their mixtures with a series of alkyl acyl carnitine esters (alkyl 3-acyloxy-4-trimethylammonium butyrate chloride) tested with CV-1 cell culture (monkey fibroblast) [127]. The influence of the aliphatic chain length (n - 12-18) on transfection in vitro was determined using cationic liposomes prepared from these lipids and their mixtures with the helper lipids DOPE and cholesterol (Fig. 30). Both helper lipids provided for significant transfection enhancements in an apparently chain-specific manner, with the highest effects found for short-chain lipids with diC12 0 and diC14 0 chains in 1 1 mixtures with the respective helper lipid. 

In this chapter, we provide an overview of our recent efforts to develop a fundamental science base for the design and preparation of optimal lipid-based carriers of DNA and siRNA for gene therapy and gene silencing. We employ synthesis of custom multivalent lipids, synchrotron X-ray diffraction (XRD) techniques, optical and cryo-electron microscopy, as well as biological assays in order to correlate the structures, chemical, and biophysical properties of cationic liposome (CL)-NA complexes to their biological activity and to clarify the interactions between CL-NA complexes and cellular components. Earlier work has been reviewed elsewhere [1-7] and will not be covered exhaustively here. 

In conclusion, we have designed a synthetic vesicular DNA carrier that physically and functionally mimics an enveloped virus particle. To achieve an acceptable degree of encapsulation within the vesicle, we use a process that is essentially inverse to the preparation of cationic lipid-DNA complexes. A suitable DNA condensing agent is introduced that, at a certain critical concentration, conveys a weak net cationic charge to the condensed DNA that then interacts spontaneously with a liposome containing one or more anionic components. These DNA formulations behave distinctly different from classic cationic liposome DNA complexes in vitro in as much as they have been shown to be nontoxic, to display a traditional linear dose response, and to be serum-insensitive. 

---