DISEÑO Y ELABORACIÓN DE LOS PERFILES DE CARGOS POR COMPETENCIAS

DNA molecules and liposome/DNA molecules are not in the range generally required for efficient gene delivery and additional condensation is therefore required. The most common view is that for efficient DNA delivery it is important for the delivery system to be o f a size between lOOnm and 200nm to achieve efficient delivery across the cell membranes. Research into DNA condensation has resulted in the use of polycations such as polyamidoamine (PAMAM) dendrimers, linear polyaspartic acid (PAA) and polymethacrylate based systems which have shown great promise and the most popular polycations, heterogeneous polylysine (PLL), defined length oligopeptides and polyethylenimine (PEI) which have demonstrated efficient DN A condensation (Bieber

et al., 2002; Rackstraw et a l, 2002). These polymers spontaneously complex with and

condense DNA, as a result o f electrostatic interactions between positively charged groups of the polycation and negatively charged phosphate groups of DNA, to form polyplexes, nanoparticles of approximately lOOnm (Gebhart and Kabanor, 2001; Jeong and Gutowska, 2002; Parkes and Hart, 2000).

The polymers that form polyplexes with DNA offer a num ber o f positive attributes to gene delivery. The linear polymers have polypeptides o f a variety of molecular

Chapter2.____________________________________________________________________Literature Survey

weights, chemical compositions, variable number o f repeating units and a varied polymer backbone architecture (linear, branched, dendritic, block or graft co-polymer). A wide range of different polyplexes can easily be synthesised and evaluated to derive the most efficient polyplex systems (Gebhart and Kabanor, 2001). Targeting signals, such as ligands for cellular or sub-cellular targeting for endosome disrupting or nuclear localising functionality, can be covalently attached relatively easily to the polymer backbone o f polyplexes (Ramsay et a l, 2000). Polyplexes have been shown to exhibit less toxicity than lipoplexes, can be easily degraded by cells and once bound to DNA the detrimental effects on cell membranes due to the high positive charge on the polymers is greatly lessened (Gebhart and Kabanor, 2001; Jones et a l, 2000; Tang and Szoka, 1997). Complexation of DNA with cationic polymers can also protect the condensed DNA from enzymatic breakdown, a crucial factor in the efficiency of gene delivery in vivo and in vitro (Jeong and Park, 2002; Schaffer et a l , 2000).

Polylysine (PLL), although not clinically exploitable has to date been the most commonly utilised model cationic polymer in laboratory gene delivery investigations, particularly for use in receptor mediated gene transfer (Jones et a l , 2000; Ramsay et a l, 2000; W olfert et a l , 1996). Like other polycations PLL interacts with the polyanionic phosphate groups of the DNA through electrostatic interactions to form discrete polyelectrolyte particles condensing the DNA to a sizes between <100- 2000nm (Figure 2.5) (Niwa et a l, 2002; W olfert et a l, 1996). The DNA/PLL complexes can attach to the cells via electrostatic interactions due to the polymers excess of positive charge interacting with the negative surface charge of the cells and facilitate DNA entry due to their net positive charge (Morpurgo et a l, 2002; Uherek and Weis, 2000). However, in most applications PLL is covalently coupled to targeting peptides to achieve improve specificity of uptake (Wasan et a l , 1999). In addition to the excellent DNA condensation properties and targeting capabilities PLL may also impart a protective effect against nuclease attack of DNA and thereby improve the eventual activity within the cell (Jeong and Park, 2002; Wasan et a l , 1999). The use of these PLL condensed complexes in vitro and in vivo applications has shown that the size of the complexes and capacity of transfection are influenced by the molecular weight o f the PLL (Wolfert et a l, 1996; Zauner et a l, 1998).

Chapter!. Literature Survey

Figure 2.5 - Poly-L-lysine Condensed pDNA

Extracellular

f

C o n d e n se d P la sm id D N A

C ell S u rfa ce R ece p to r

Intracellular

K ey: L ig an d P oly-L -lysine

T h erap eu tic G ene In sert

Adapted from Smith and Doherty, 2000.

A lth o u g h the D N A co n d e n se d by p o ly c a tio n s is o f an ideal size fo r tran sfectio n there

are few ex a m p le s o f such fo rm u la tio n s w ith tran sfectio n e fficien cie s in vivo su p erio r to

n aked D N A . O ne reason is that th ey are in trin sic ally a g g re g a tin g an d gen erally ag g reg ate in p h y sio lo g ical c o n d itio n s an d form en titie s to o large to p enetrate

e ffe c tiv e ly th ro u g h solid tissu e s an d to be tak en up by m o st cell ty p es (Jones et a l ,

2000; M o u n tain , 2000; P o u to n et a l , 1998). A sec o n d lim itatio n o f th e use o f cationic

p o ly m ers as tran sfectio n agen ts is th ere d etrim en tal in flu en c e on cell viability. A c co rd in g to m an y au th o rs P L L is c y to to x ic , p ro b a b ly in v o lv in g cell m em brane

Chapter!.____________________________________________________________________Literature Survey

damage due to interactions between the negative charged membrane components and PLL cationic side chains. However, there is contradictory evidence from authors such as Geghar and Kabanov (2001) who reported that lipoplexes were found to be more toxic than polyplexes and there was no significant toxicity after cells were exposed to free polycations. Furthermore PLL is already used as a component of the microencapsulation system used to protect live cells in allogeneic transplantation from immune attach, indicating that PLL antigenicity should not be o f concern (Wasan et a l, 1999). Having identified a number of promising cationic polymer candidates to condense DNA to a size that does not limit gene delivery efficiency further investigation is required in order to circumvent the aggregative and toxic properties and of these vectors and it is likely that the attention will now be shifted to the incorporation of polycations into multi-component systems in order to improve their efficacy (Rackstraw et a l , 2002).