For in vivo applications, the development of polyplexes is not only confronted with intracellular barriers, but also with extra cellular barriers, which are those prior to reaching the target cells. A schematic diagram of the potential intracellular and extracellular fate of a polyplex is shown in (Fig. 3). Strategies have to be developed in designing polyplexes that enable them to survive blood and other biological fluids, and to escape extracellular physical barriers, in order to reach the target cells. The specific strategies must take into account the physicochemical properties such as size, shape and flexibility, overall charge, charge density, and nonelectrostatic interactions at the surface of the polyplex. Besides considering these physical properties, one could imagine to also use active endogenous cellular transport mechanisms, such as transcytosis (12,144).
In vitro gene delivery and ex vivo gene therapy approaches are predominantly concerned with gene delivery barriers presented by the cell itself. In vivo gene therapy must also be concerned with extracellular barriers, where the route of administration of the therapeutic gene also plays a major role (145). In systemic administration, where the vector is introduced in the body intravenously, the circulatory pathway and environment, as well as various nontarget cells and organs, encountered by the polyplex are major obstacles, determining the fate of the transferred DNA [see, for example, (146)]. However, local administration methods, such as direct injection of the vectors into the target region, are not confronted with the circulation problem, but nevertheless are still confronted with barriers such as extracellular matrix or inflammatory and immune responses.
A. Physical Restrictions of Transfection Particles
Size seems to be a general critical factor for drug targeting
(147). Because of size restriction, (several) hundred nm large particles cannot penetrate endothelial and epithelial barriers
(148), or extravasate from the vascular to the interstitial space. Particle size is also an important factor when considering organ clearance and intraorgan distribution. For example, particles that are too large to pass through the vascular endothelium to the liver parenchym are engulfed and degraded by liver Kupffer cells (i.e., phagocytic cells residing next to the vascular epithelial cells).
DNA is a negatively charged, flexible molecule, both characteristics that hinder the DNA from being transported efficiently into and across cells, which in this case represent the physical barriers. To overcome these physical drawbacks, the DNA molecule needs to be compacted and the negative charges minimized for efficient gene transfer. These requirements can be achieved by taking advantage of molecules capable of binding and condensing DNA (149). The structure of condensed DNA complexes has been analyzed in several reports, such as (13,150-152,14). DNA-polycation complexes have been characterized by electron microscopy and atomic force microscopy (shape, size), laser light scattering (size), electrophoretic mobility (reflects charge and size of complexes), zeta potential measurements (charge), circular dichroism (conformation of DNA), or centrifugation techniques (molecular weight and condensation). The results give some insight on how to possibly generate DNA complexes sufficiently small to traverse the en-
dothelial layer, possibly through fenestrations or vesicular transport systems, such as transcytosis (Fig. 4). In addition, the compact DNA complexes are more stable against enzymatic or mechanical degradation, which may take place during DNA transport processes to the target cells/tissue.
However, preparation of effective DNA complex molecules for in vivo delivery of DNA remains a major hurdle. The extent of DNA condensation depends on a number of variables, including the ratio of positively charged DNA-binding element (''cationic carrier'') to negatively charged DNA, the size and modification of the DNA-binding element, size, sequence, and state of the DNA, and also the procedure of complex formation (29), strongly influencing the in vitro and in vivo gene transfer efficiency. The net charge of the DNA-cationic carrier complex affects its solubility. Complexes with either an excess of DNA or positively charged carrier are stabilized in solution by the negative or positive charges. At molar charge ratios [see(1): positive charges of carrier to negative charges of DNA phosphates] close to 1 (i.e., electroneutrality), hydrophobic domains of DNA-binding elements such as polylysine are considered to be responsible for low solubility in water. This may lead to aggregation and precipitation of complexes.
Methods of formulation still have to be improved in generating homogenous and stable complexes capable of overcoming the physical barriers. DNA-polylysine-conjugate complexes have been prepared in several ways. Wu and Wu (40) mixed the compounds at high salt concentration, where electrostatic binding is strongly reduced. Slow reduction of the salt concentration by dialysis into physiological buffer results in a thermo-dynamically controlled complex formation. Charge ratios of polylysine/DNA smaller than 1 and enhanced hydrophilicity due to the conjugated asialoglycoprotein are presumably essential for the solubility of the complex.
Wagner et al. (13) described a different approach using transferrin-polylysine-DNA complexes. Flash mixing of dilute compounds in physiological phosphate-free buffer results in formation of kinetically controlled complexes. Charge ratios of polylysine/DNA from smaller than 1/2 to larger than 2/1 have been applied. At ratios of electroneutrality or higher, donutlike and rodlike particles of 80 to 120 nm in diameter are formed.
Complexes containing transferrin-conjugated polylysine have increased solubility compared with the use of unmodified poly-lysine.
Interestingly, donuts of similar sizes are formed, independent from whether small or large (up to 48 kbp) and DNA is used in the complex formation. Using a standard expression plasmid of approximately 5 kbp, obviously several DNA molecules are incorporated into 1 particle. In an attempt to generate unimolecular DNA complexes, Perales and colleagues (35,60) added polylysine conjugates slowly, in several small portions, to a vortexing solution of DNA in approx. 0.5 to 0.9 M sodium chloride until a charge ratio of polylysine/DNA of approximately 0.7 is reached. The slow addition of polylysine has been reported to generate monomeric DNA complexes, with sizes of approximately 15 to 30 nm. These complexes aggregate immediately; aggregation is reverted by subsequent addition of salt. The promising findings have been reported to be applicable for in vivo gene transfer applications (35,60).
Recent reports on the size of DNA complexes with PEI or transferrin-PEI describe the strong influence of parameters such as DNA concentration and charge ratio, and also ionic strength of solution, or serum content of culture medium. Mixing DNA-PEI complexes at N/P (PEI nitrogen: DNA phosphate) molar ratios below 6 in 150 mM saline results in rapid aggregation; aggregation can be avoidedby complex formation at low ionic strength (25 mM aqueous buffer), generating particles with an average diameter of approximately 40 to 50 nm (14). Incorporation of hydrophilic polyethylene glycol residues into polyplexes also was found to stabilize small polyplexes and prevent their aggregation (153).
B. Undesired Interactions with Plasma, Degradative Enzymes, Matrix, and Nontarget Tissue
Polyplexes, when administered in vivo, are surrounded by a variety of compounds present in blood plasma. Salts, lipids, carbohydrates, proteins, or enzymes contribute to changes in the physicochemical properties of the polyplex. Some of these factors (''opsonins'') may coat the polyplex, causing aggregation, dissociation, or degradation of the DNA complex. This may influence the composition of the complex as well as the bioavaili-bility. Thus, the DNA complexes, even when reaching the target cells/tissue, may no longer exhibit the physical properties necessary for efficient transfer into cells.
Previous studies have demonstrated the inactivation of poly-lysine-based DNA complexes by blood components (154). One of the factors was identified as the complement system (155). More recently, the interaction of DNA-PEI complexes with plasma was analyzed on the biochemical level. Upon incubation of the DNA complexes with human plasma, specific proteins (IgM, fibrinogen, fibronectin, and complement C3) bind to the complexes (153). By coating the DNA-PEI complexes with polyethylenglycol (PEG) through covalent coupling to PEI, plasma protein binding was found to be strongly reduced (153).
Another problem encountered in the bloodstream is degra-dative enzymes. There are nucleases in the bloodstream that degrade extracellular DNA (such as that generated by degradation of invading microorganisms or dead host cells). Cationic DNA-binding elements may serve some protection (45).
Other undesired interactions are the ones with the extracellular matrix and nontarget cells/tissue. There is a complex network of proteins and proteoglycans, termed extracellular matrix that fill the intercellular space (156,157). The matrix helps bind the cells in tissues together and also provides a lattice through which cells can move. Once the DNA complex has traveled across the vascular barrier into the interstitial space, it has to avoid interactions with the extracellular matrix in order to reach and bind the target cells/tissue. Extracellular matrices in animals are composed of different combinations of collagens, proteoglycans, hyaluronic acid, fibronectin, and other glycoproteins. These components could serve as specific barriers by binding the DNA complexes. For example, hyaluronic acid binds cations very effectively. With this in mind, DNA condensed by polycations, resulting in a net-positive charge, could also interact with the extracellular matrix, binding, dissociating, or aggregating the DNA complex. Thus, proper formulations, in preparing the DNA complexes, will be necessary, to avoid such interactions. Optimizing the DNA complexes bearing an overall low-charge ratio close to neutrality is a possibility to avoid such interactions. The problem is that a net-positive charge has been found desirable for interaction with the cellular plasma membrane and entry into the target cell, whereas such positive charge might favor entrapment of the DNA complexes by negatively charged extracellular matrix components.
Interaction with nontarget cells/tissue is another hurdle, complicated to combat. In the previous sections, cellular inter-nalization mechanisms using receptor-specific ligands were presented as a solution to overcome this problem. Ligands may target cells very efficiently, but it has to be kept in mind that they do not inhibit unspecific interactions with nontarget cells, which could result in cellular binding and internalization via any additional process. The unspecific interactions with nontarget cells may be due to factors such as particle size, charge, and in vivo protein coating of the DNA complexes. For example, interaction with nontarget cells may take place due to an excess positive charge of the complex. It has been shown in cell culture that minor changes in the DNA/polylysine-conjugate ratio of the complex, resulting in a positively charged DNA complex, may convert a ligand-specific transfer into a completely unspecific process (29). Ideally, the DNA complex should be masked in a fashion that only allows ligand-receptor interactions with the target cell, and no other interactions with nontarget cells.
As a result of introducing foreign molecules into the body, individual immune cells are stimulated to produce antibodies, a process termed humoral immunity. In addition to this humoral response, specific T cells may also be activated (cellular immunity). These 2 processes are the specific immune re sponse. There is, however, also the nonspecific immune response, including phagocytosis, inflammation, and other nonspecific host-resistance mechanisms such as the complement system (158). These nonspecific mechanisms develop immediately against virtually any foreign molecule, even those the host has never encountered. Thus, the nonspecific immune response is a major extracellular barrier for the DNA complex, which in this case is the foreign molecule. The ultimate goal is to formulate and construct polyplexes in a manner, that avoid eliciting any immune response.
Inflammatory response is a major problem for any gene delivery system (159) because it may take place independent from the route of administration and results in a greater access of phagocytes to the foreign molecules, for example, due to an increased capillary permeability caused by retraction of the endothelial cells. During an inflammatory response, leucocytes, particularly neutrophil polymorphs and to a lesser extent macrophages, migrate out of the capillaries into the surrounding tissue. At the site of inflammation, thephagocytes recognize the foreign molecules via receptors on their surface, which allow them to attach nonspecifically and phagocytose-foreign molecules. Attachment is greatly enhanced and specified upon opsonization of foreign molecules, such as by the C3b component of complement. Both neutrophils and macrophages have receptors that specifically bind to C3b, allowing them to recognize their target.
A variety of macromolecules, such as proteins, lipoproteins, some nucleic acids, and many polysaccharides, can act as immunogens under appropriate conditions. Positively charged DNA complexes have the ability to activate the complement system (155). A number of synthetic cationic molecules, frequently used in gene delivery, and their complexes with DNA have recently been examined for their complement-activating properties. Complement activation by polylysine is strongly dependent on chain length and on the charge ratio. Longer chains and greater surface charge density are strong activators of the complement system. The positive charges on the DNA complex are accessible to the complement protein C3b. Opsonization of such particles by C3b leads to the initiation of a cascade of events, presumably resulting in the clearance of DNA complexes by the retinoculoendothelial system. Coating of the positive charges of the DNA complexes with other macromolecules may inhibit interactions with components of complement, hence decreasing complement activation and clearance of the complexes from the blood circulation (160). It has already been demonstrated that modification of the surface of liposomes reduces interaction with blood components (161-163), stabilizes DNA-liposome complexes (164). Coating of polycat-ion-DNA complexes by polyethyleneglycol (PEG) also reduces interaction of DNA complexes with blood components. (153,155).
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