Biological barriers to cellular delivery of lipid-based DNA carriers

Advanced Drug Delivery Reviews 38 (1999) 291–315

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Biological barriers to cellular delivery of lipid-based DNA carriers Marcel B. Bally*, Pierrot Harvie, Frances M.P. Wong, Spencer Kong, Ellen K. Wasan, Dorothy L. Reimer Medical Oncology– Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10 th Avenue, Vancouver, British Columbia V5 Z 4 E6, Canada

Abstract Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure / activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.  1999 Elsevier Science B.V. All rights reserved. Keywords: DNA transfer; Transfection; Cationic lipids; Gene therapy

Contents 1. 2. 3. 4. 5. 6.

Foreword ................................................................................................................................................................................ Introduction to the technology .................................................................................................................................................. Defining the ‘activities’ of lipid-based plasmid delivery systems................................................................................................. Preparation of lipid–DNA complexes ....................................................................................................................................... The first barrier: protection of the plasmid expression vector ...................................................................................................... The second barrier: binding to the cell membrane ......................................................................................................................

*Corresponding author. Tel.: 1 1-604-877-6098, extn. 3191; fax: 1 1-604-877-6011. E-mail address: [email protected] (M.B. Bally) 0169-409X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 99 )00034-4

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6.1. Regional administration .................................................................................................................................................... 6.2. Designing lipoplexes for intravenous applications............................................................................................................... 6.3. Target specific binding ..................................................................................................................................................... 6.4. Cell binding, delivery and transfection efficiency: is there a correlation? .............................................................................. 7. The third barrier: DNA delivery and dissociation from the carrier ............................................................................................... 7.1. Membrane destabilization reactions ................................................................................................................................... 7.2. Dissociation of the lipid / DNA complex............................................................................................................................. 7.3. Endosomal lysis and other modes of escape ....................................................................................................................... 8. The fourth barrier: transfer into the nucleus ............................................................................................................................... 9. Summary – lipoplex design challenges ..................................................................................................................................... Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................

1. Foreword The challenge that one faces when writing a review is to identify a focus. This is a difficult task in the area of gene therapy because the technologies and therapeutic applications are so diverse. A brief search of the MEDLINE data base of scientific literature indicated that over 300 reviews on gene therapy were published over a 9 month period ending in September 1997, the time when this review was prepared. A cursory examination of these reviews leaves one with the feeling that researchers in this field are more enamoured by the therapeutic potential of controlling gene expression within cells or tissues then by the mechanics of how this control will be achieved. This is best exemplified by studies examining the use of plasmid expression vectors for gene transfer, where transgene expression is the standard by which the technology is currently being judged. There has been a great deal of effort evaluating how plasmid design and plasmid DNA delivery technology affects transgene expression. In contrast, very little effort has been focused on plasmid transfer into and through cells, a principal requirement for transfection to occur. It is still not clear, however, whether plasmid delivery is sufficient for gene expression or whether the delivery technology plays a role in plasmid expression following delivery. This review will focus on plasmid transfer systems and, in particular, on the role of lipid-based carriers used to achieve delivery of plasmid expression vectors. We will not consider specific issues regarding design of plasmid expression vectors or the cationic lipid chemistry and synthesis. For the purpose of this review we refer to plasmids in a generic context. In terms of plasmid delivery there is little

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evidence suggesting that a 5 kb plasmid containing the chloramphenicol acetyltransferase gene behaves differently than an 8 kb plasmid containing the b-galactosidase gene. In terms of transgene expression, this is a naive assumption considering that regulation of b-galactosidase expression may be quite different from chloramphenicol acetyltransferase expression even when the same promoter is used to drive expression of the different genes. We also refer to cationic lipids in a generic context, where the primary role of the lipid is to facilitate electrostatic interactions between the lipid or liposome and DNA in order to generate the carrier system. Given the number of cationic lipids available for gene transfer purposes, an assumption suggesting that all cationic lipids behave in a comparable fashion is also easily criticized. We will define the carrier in terms of the cationic lipids required for DNA binding, additional lipids required for control of carrier stability and the plasmid DNA as the active agent. This issue of Advanced Drug Delivery Reviews is concerned primarily with how membrane destabilization may bring about intracellular delivery and subsequent transfer of DNA to the nucleus. It is, however, difficult to focus on these two events without consideration of how the delivery system is designed to achieve protection of the therapeutic agent and cell delivery. As demonstrated for conventional drugs, it is relatively easy to design carrier systems suitable for cell delivery in vitro, but these systems often prove to be unsuitable for parenteral administration. What will be reviewed here are the efforts from our laboratory and others characterizing the steps involved in gene delivery and the biological barriers that are encountered when crossing these steps. The steps and barriers that we believe are

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Fig. 1. The six steps and associated biological barriers that must be crossed in order for a gene transfer system to be therapeutically useful. Progression through the first four steps (DNA protection, access and binding to the target cell population, internalization and DNA release within the cytoplasm of the cell) is dependent on the attributes of the carrier formulation. The remaining two steps, transgene expression and therapeutic response, rely on characteristics of the plasmid expression vector and the gene being expressed.

important are shown in Fig. 1. We strongly believe that further advances in the use of plasmid expression vectors for gene therapy is critically dependent on designing delivery systems and plasmid expression vectors capable of overcoming all the barriers outlined.

2. Introduction to the technology Recent advances in molecular genetics have contributed significantly to our understanding of disease pathogenesis [1,2]. A logical extension of this new understanding concerns the development of therapeutic strategies that specifically target genetic abnormalities [3,4]. Advances in molecular biology have resulted in a new concept for treatment of disease, the concept of gene therapy. Gene therapy can be defined as any treatment strategy that relies on the introduction of polynucleotides into human cells for the purposes of reduction or elimination of disease. Polynucleotides would include antisense oligonucleotides [5,6], ribozymes [7,8] as well as DNA in a viral or plasmid vector [9–12]. Clearly, the ability to successfully introduce nucleic acid sequences and DNA into cells has brought to light a new approach for the treatment of many human diseases. The technology will potentially impact all diseases presently affecting humans including cancer [13], inflammatory diseases [14], infectious diseases [15], vascular disease [16], neurological disorders [17] as

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well as inheritable genetic abnormalities [18,19]. The concept is simple and involves the delivery of nucleic acids to target cells in an effort to alter the production of a specific protein, where changes in protein expression result in a therapeutic benefit. Similar to other therapeutic strategies involving administration of biologically active molecules, the success of gene therapy is dependent on efficient delivery and expression of the therapeutic agent to target cells within diseased tissues or organs. Unlike conventional small molecules, however, polynucleotides exhibit chemical and physical properties that are not well suited to cell delivery. In an effort to achieve efficient polynucleotide transfer, a variety of delivery systems have been developed. These delivery systems have one purpose: to improve the therapeutic activity of the polynucleotide. This is an important point that clearly separates the role of the delivery technology from the role of the therapeutic agent that may require use of a carrier for activity to be expressed. It is now well established that antisense molecules, either antisense oligonucleotides or ribozymes, are therapeutically active in vivo and this activity is observed in the absence of a specific carrier formulation [20,21]. To improve the therapeutic properties of these molecules, it may be sufficient to develop delivery technology that protects and delivers the antisense molecules to the region where the target cell is localized. Further improvements may then be achieved by targeting the antisense carrier formulation to a defined cell population and / or by augmenting intracellular delivery. Ultimately, it may be possible to design delivery systems that transfer the antisense molecules to a specific intracellular compartment where optimal activity can be obtained. As indicated above, one can outline a sequential and ordered optimization plan for the use of carrier technology to improve the therapeutic activity of antisense molecules. This, however, is more difficult to envision for plasmid expression vector delivery systems. Although it has been shown that plasmid alone can transfect muscle cells, a fact that has been exploited in the development of vaccines [22], it is generally assumed that plasmid expression vectors will require a carrier in order to obtain efficient gene transfer to the nucleus. Nature’s vector is the virus and recombinant viruses are at the present time the

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most efficient way to deliver genes to cells [23,24]. Plasmid vectors are available and interest in developing this technology is a result of concerns about the safety of viral vectors [25] and limits in the viral insert size. In general, plasmid expression vectors must be formulated in a manner that protects the DNA from nucleases. In addition, the physical and chemical properties of the DNA formulation must facilitate transfer of the plasmid into cells. In contrast to carrier systems for antisense molecules, we believe that it will not be sufficient to simply achieve plasmid delivery to the region where the target cells are located. Standard methods used to formulate plasmid DNA for gene transfer rely on polycations to complex the DNA. It has been demonstrated that cationic proteins (e.g., histones) and polylysine can condense DNA [26,27]. Condensation of DNA has been characterized extensively by Bloomfield [28,29] and is of basic interest for those attempting to elucidate the physical characteristics of DNA in viruses, bacteria and eukaryotic cells. The importance of DNA condensation in methods involving gene transfer has yet to be determined, although it has been shown that condensed DNA is inaccessible to small molecules, such as the DNA intercalating dye ethidium bromide, and is protected against degradation by DNase I [30]. As indicated in Section 5, such protection is required following parenteral administration of the plasmid DNA. Studies evaluating the physical characteristics of condensed DNA suggested that for DNA ranging in size from 0.4 to 48 kb, condensation yields a donut-like toroidal structure that has a uniform size of 40 to 60 nm. These particles may contain 1 to 30 DNA molecules per particle depending on the size of the DNA molecule used. Formation of the toroid structures is considered energetically favorable and occurs spontaneously after 90% of the DNA phosphate charge has been neutralized. Structures similar to those of condensed DNA have been obtained using a variety of polycations, alone or in combination. This includes: (i) monovalent and polyvalent cationic lipids formulated as liposomes [31,32] or as hydrophobic lipid–polynucleotide complexes [33–35], (ii) polycationic polymers [36,37] and (iii) polycationic peptides [38,39]. The structures obtained using these polycations to complex DNA are variable and depend on factors

that are too often not clearly understood. Regardless, almost all formulation technologies have proven to be effective at achieving gene transfer as judged by transgene expression in selected cells in culture or certain tissues following parenteral administration. The expression levels are low, however, and it will be important to understand mechanisms of delivery if expression is to be enhanced to the level required to observe therapeutic activity. Attempts to relate the physical and chemical attributes of these delivery systems to the levels of expression obtained have not been successful to date. This is partially due to the fact that changes in one component used in the formulation often affects more then one attribute of the macromolecular structure adopted by the carrier. This, in turn, has made it difficult to prove structure / activity relationships.

3. Defining the ‘activities’ of lipid-based plasmid delivery systems In order to develop useful plasmid delivery systems, the carrier formulation must fulfill many ‘activities’ in order to facilitate transgene expression. If the activity of the delivery system is defined in a narrow context, then one can judiciously address how individual components of the delivery systems affect specific requirements, such as protection of the associated DNA. Although six steps have been defined in Fig. 1 as requirements for a therapeutically useful gene transfer system, in the case of lipid-based carriers we believe that only the first four are dependent on the attributes of the carrier formulation. The remaining two steps, transgene expression and therapeutic response, rely on characteristics of the plasmid expression vector and the gene being expressed as well as the biological status of the cell. These two steps will not be discussed further in this manuscript. Based on the first four requirements, we would suggest that the carrier system must exhibit certain attributes in order for each biological barrier to be crossed. Importantly, the role of the formulation’s physical and chemical attributes can be determined with respect to the individual barriers. We believe that it is important that the biological barriers be studied in isolation; recognizing, however, that the

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functional roles must act in concert in order to achieve efficient cytoplasmic delivery. It may, for example, be relatively easy to develop a serum stable plasmid formulation that is not capable of being internalized by a target cell. Conversely, an efficient fusogenic delivery system that facilitates cytoplasmic delivery of plasmid expression vectors may be destroyed readily when incubated in serum. We believe that there are four critical events required for plasmid delivery to occur. First, the active agent (the plasmid expression vector) must be protected from degradation by nucleases in the tissue culture media, serum and sites of injection or interstitial spaces. Second, the lipid–DNA complex must come in contact with the target cell. For in vivo applications, access to the target cell will be dependent, in part, on the route of administration. Third, the carrier system must interact with the target cell membrane and subsequently undergo internalization or cytoplasmic delivery. We believe that, in addition to achieving membrane destabilization, cytoplasmic delivery also requires dissociation of the lipid components from the plasmid. Fourth, the transfected DNA must cross the nuclear membrane and be processed as required for efficient transcription and translation. The focus of the remaining sections of this article will be to review what is known about cellular and molecular mechanisms involved in the processing of lipid-based DNA carriers with the aim of identifying specific methodologies that can be used to overcome barriers to cellular delivery. In order to do this a summary of current technology used in preparation of lipid-based plasmid delivery systems is useful.

4. Preparation of lipid–DNA complexes The concept of using lipid-based carriers for delivery of DNA to cells resulted from an extensive amount of research on the use of liposomes as drug carriers [40]. As DNA carriers, this technology relied on capturing DNA within the aqueous compartments of multilamellar liposomes during their formation; a simple process requiring hydration of dried lipids with a solution containing DNA. The liposomes were typically prepared using neutral and / or anionic lipids and under ideal conditions these liposomes would

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exhibit trapped aqueous volumes of between 1 and 5 ml / mmol lipid [41]. As a consequence of the size, charge and structure, DNA does not behave like small water-soluble markers typically used to define a trapped volume and the efficiency of DNA encapsulation was typically very low. Regardless, these initial formulations could be used for gene delivery [42–44]. Improvements in the association efficiency as well as transfection activity of liposome / DNA formulations were achieved through the use of positively charged liposomes, a procedure that was first reported in 1987 [31,32,45]. Since this report, cationic liposomes have become one of the most widely studied and widely used methods for accomplishing delivery of plasmid expression vectors. Cationic liposome–DNA complexes are produced by simply mixing cationic liposomes, typically prepared in non-ionic solutions, with a solution of DNA. Positively charged liposomes bind the negatively charged phosphate molecules on the DNA backbone through electrostatic interactions, forming a complex between the liposomes and the DNA. Although complex formation provides an efficient way to associate DNA with a liposome, the binding reaction is driven by interactions between two polyvalent surfaces. As shown in Fig. 2 (liposome DNA aggregates), such multivalent binding reactions typically result in aggregation of the components [46,47]. Aggregation due to liposome–DNA–liposome crosslinking is extremely difficult to control, although the size of the aggregate can be minimized by decreasing the concentration of the reactive groups and / or the ratio of charged groups. Cationic liposomes used for gene transfer are typically small ( , 100 nm) prior to addition of DNA. The aggregate structures obtained after mixing exhibit mean particle diameters ranging from as small as 200 nm to structures larger then 2 mm [48]. The aggregation problem is further aggravated depending on: (i) the cationic lipid used, (ii) the type and quantity of additional lipids (e.g., dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC) or cholesterol) used to prepare the cationic liposome, (iii) the composition of the diluents and buffers used, (iv) the method and rate of mixing, (v) the temperature of mixing, (vi) DNA purity and (vii) the length of time after complex formation. Although cationic liposome technology has ad-

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Fig. 2. Lipoplex [55] formulation technology and hypothetical structures. It is suggested that lipid-based DNA complexes can be prepared by mixing DNA with pre-formed cationic liposomes (left panel). This initiates an aggregation reaction and formation of a heterogeneous group of structures. A careful physical characterization of a cationic liposome–DNA complex structure led Radler et al. to propose a novel multilamellar structure with DNA sandwiched between lipid bilayers [49]. An alternative formulation procedure, illustrated in the diagram on the right panel, has been described where a hydrophobic complex is used as an intermediate in the preparation of lipid–DNA particles [35,50]. The approach relies on the generation of mixed micelles containing detergent, cationic lipid, and selected zwitterionic lipids. Under appropriate conditions cationic lipid–DNA complexes, prepared in detergent, spontaneously form intermediate structures which may consist of either monomeric lipids and detergent or mixed lipid–detergent micelles bound to the DNA. Upon dialysis to remove the detergent, small particles ( , 150 nm) form. A generic lipoplex structure has been proposed by Dr. P. Felgner which depicts plasmid coated or ensheathed with lipids to form a compact condensed structure [45].

vanced to the stage where it is being tested in gene therapy clinical trials, the ability to formulate liposome–DNA complexes remains unpredictable. We and others believe that variability in the ability to generate well-defined macromolecular structures contributes significantly to difficulties in predicting the effectiveness of these formulations [49,50]. A better understanding of how DNA interacts with liposomes and lipid components will allow meaningful structure–function relationships to be established.

Such research has identified a range of membrane structures including small vesicular shapes [50,51], amorphous fused structures [51], extended tubes of DNA surrounded by a lipid bilayer [52] and / or novel multilamellar structures consisting of sheets of DNA molecules sandwiched between lipid bilayers [49]. It is unclear whether methods that use pre-formed liposomes can be devised such that one structure predominates. Until this can be done it will be difficult to determine which structure is most important for supporting transfection. In an effort to avoid the use of pre-formed liposomes, our laboratory has developed a formulation approach that relies on generation of a hydrophobic cationic lipid / DNA complex intermediate (Fig. 2, lipid DNA particles) [33–35,50]. A fortuitous observation led to the conclusion that cationic lipids could bind to DNA in the absence of a defined membrane structure. Binding resulted in the formation of a complex that was hydrophobic and could be readily isolated in organic solvents [33]. We have shown that the hydrophobic lipid–DNA complex can be prepared in the absence of pre-formed liposomes and that other lipids can be present during complex formation [34]. Importantly, complex formation was dependent on having a cationic lipid to DNA phosphate ratio of 1 or greater, implying that charge neutralization was required. We have characterized the binding reactions that lead to formation of the hydrophobic complex, studies that suggested that the binding reaction occurs at an organic / aqueous interface in a two-phase extraction system [34]. We concluded that the binding reaction follows a sequence of events involving extraction of lipids into an organic phase, followed by formation of a charged interface between the aqueous and organic phase. This interface consists of cationic lipids oriented with their headgroups facing the aqueous phase and their acyl chains maintaining maximum contact with the organic phase. DNA binds to the lipids after formation of this charged interface. When sufficient lipids are bound to the DNA the resulting complex exhibits hydrophobic characteristics and the complex can be extracted into the organic phase. This binding reaction is the result of cooperative electrostatic interactions between a multivalent anion and a multivalent monolayer of cationic lipids oriented at the aqueous / organic interface.

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Formation of hydrophobic cationic lipid / DNA complexes is of interest in terms of developing a better understanding of the chemical and physical attributes of novel self-assembling structures. In addition, this complex can be used as a unique intermediate in the preparation of lipid-based carrier systems for plasmid expression vectors [35,50]. The lipid–DNA complex, in the absence of stabilizing factors, can adopt unique macromolecular structures suitable for use as a DNA carrier. For example, the complex can be isolated in organic solvents and following solvent removal the complex can be solubilized in oils to be used in the preparation of oil / water emulsions [53]. Alternatively the complex can be solubilized in organic solvents appropriate for use in the preparation of membrane structures via application of reverse-phase evaporation techniques or by ethanol injection. We have placed emphasis on structures formed from lipid–DNA complexes prepared in non-ionic detergents [35]. The method consists of preparing mixed micelles containing the detergent, cationic lipids and other lipids as required for optimizing plasmid delivery. Subsequently, these micelles are combined with plasmids, also diluted in detergent. The concentration of detergent used in preparing these formulations is critical and plays a significant role in controlling the physical properties of the particulate carriers generated following formation of the cationic lipid–DNA complexes [54]. When the detergent concentration is close to its critical micelle concentration (CMC), inter- and intra-molecular forces between lipids bound to the DNA drive spontaneous formation of a particulate structure. The characteristics of the particles formed using this procedure have been described in detail elsewhere [35,50,54]. There are two important features of these lipid-based DNA formulations that are worth emphasizing. Under appropriate conditions the structures which form are small ( , 120 nm). The size of the structure is critically dependent on the charge ratio (cationic lipid to DNA phosphate ratio), detergent concentration, the type and amount of noncationic lipid present during particle formation as well as the buffer composition. However, these parameters can be easily determined and a variety of lipid compositions can be used in the preparation of uniformly small plasmid delivery systems [54]. The

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second important feature, as shown in Fig. 3, is that certain formulations are as effective as those prepared using cationic liposomes in terms of transfection efficiency. Other researchers working in Sullivan’s laboratory have also generated stable, transfection competent, macromolecular lipid-based DNA formulations using detergents as media in which to prepare the formulations [78]. We believe that the self-assembling structure described above represents a new class of polymeric delivery system. The delivery system relies on particle formation that is a consequence of cationic lipid binding directly to an anionic polymer. In the

Fig. 3. Lipoplex-mediated delivery of a plasmid expression vector incorporating the gene for chloramphenicol acetyl transferase (from Ref. [54]). Murine B16 / BL6 melanoma cells were plated at 4000 cells / well in a 96-well plate containing media supplemented with 10% fetal bovine serum and grown overnight. Lipoplex formulations prepared using the hydrophobic cationic lipid–DNA complex intermediate were composed of the cationic lipid N,Ndioleoyl-N,N-dimethylammonium chloride (DODAC) and various helper lipids such that the cationic lipid to DNA phosphate ratio ( 1 / 2 ) was 2:1 and the neutral to cationic lipid molar ratio was 1:1. Lipoplexes were prepared using DOPC (1), DOPE (2), DLPC (3), or DLPE (4) as the added helper lipid. Transfection was compared to free DNA or lipoplexes prepared using pre-formed cationic liposomes (DODAC / DOPE, 1:1). DNA formulations were added and incubated in serum-containing media for 4 h. Media were removed and replaced with fresh media for a further 48 h and chloramphenicol acetyltransferase (CAT) activity was evaluated. Values were determined from three replicates and expressed as mean6standard error of the mean.

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presence of detergents the self-assembly process likely consists of additional conformational changes involving both DNA and lipid. The illustration shown in Fig. 2 suggests that cationic lipids solubilized in mixed lipid–detergent micelles may crosslink DNA molecules. Alternatively, we propose that cationic lipid binding to DNA is dependent on an equilibrium reaction between cationic lipid associated with micelles (one phase) and that bound to DNA (an alternative phase). Regardless, the ability to control lipid composition while maintaining comparable particle size is one of the primary advantages of this new formulation approach. We have used formulations prepared by this new approach as well as those prepared using conventional cationic liposome / DNA aggregates to characterize how formulation parameters, such as lipid composition, control cell binding and intracellular delivery of DNA. This effort is in no way unique, since many groups are focused on elucidating the role of carrier attributes in defining cell delivery, each relying on formulation procedures developed in their own laboratory. For this reason, we will refer here to the structures adopted by the different formulation techniques as ‘lipoplexes’, a name recently coined by researchers who have been instrumental in the development of lipid-based plasmid delivery systems [55]. A hypothetical structure of a lipoplex has been proposed by Felgner [45]. Regardless of what approach is taken to prepare these particles, it can be suggested that such a structure is well-suited for plasmid delivery. The properties of these carrier systems can be effectively manipulated to achieve formulations that are stable following parenteral administration and can be modified appropriately to achieve target-specific cell delivery as well as effective intracellular delivery of the associated plasmid expression vectors.

5. The first barrier: protection of the plasmid expression vector The physical characteristics of the lipoplexes being developed for delivery of plasmid expression vectors must protect the associated DNA from factors that will prevent transgene expression. Superficially, lipid-mediated protection of plasmid expres-

sion vectors is easy to measure, easy to achieve and most approaches used to prepare lipoplexes result in some level of DNA protection, as assessed by inhibition of DNA intercalating dye binding and DNA stability in the presence of DNase I or serum [56]. The dye-binding assays typically rely on the use of intercalating agents that fluoresce when bound to DNA [57]. It is known that condensed DNA, prepared by mixing polylysine with DNA, is in a form that does not allow dye binding to occur. Similar results are obtained for lipoplex-formulated DNA, where dye exclusion may be due to changes in DNA structure or the presence of a bound monolayer or bilayer of associated lipid. It is unclear, therefore, whether dye exclusion is a consequence of DNA condensation or dye displacement by cationic lipid– DNA binding. For example, as shown in Fig. 4, lipid binding can generate hydrophobic cationic lipid / DNA complexes that can still bind the intercalating dye TO-PRO-1. In addition, data from our laboratory have demonstrated that cationic lipid-mediated particle formation in the presence of detergent can yield a macromolecular structure that permits dye binding yet still maintains DNA in a form that is protected from DNase I degradation [35]. As indicated in Section 7.2, DNA intercalating dye-binding assays can also be used to measure dissociation of lipids from DNA and it can be used to assess lipoplex formulation characteristics under more physiological conditions such as in the presence of salts and serum proteins [58]. This is illustrated in Fig. 5. These data demonstrated that plasmid DNA was protected equally, in terms of TO-PRO-1 exclusion, for a variety of lipid-based DNA formulations. Dye exclusion indices, a parameter that measures dye binding relative to that observed using polylysine condensed DNA, were greater than 95% regardless of the lipoplex lipid composition or the method employed to prepare the lipoplex. For reference, an exclusion index approaching 100% is observed for polylysine condensed DNA. Importantly, addition of salts (150 mM NaCl) and 10% serum leads to a rapid ( , 1 min) and significant decrease in the dye exclusion index for all DNA formulations studied. In general, the addition of salt caused the dye exclusion indices to decrease from . 95% to values between 40 and 70%. Salt-

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Fig. 4. Evaluation of DNA accessibility assayed by TO-PRO-1 dye intercalation (from Ref. [33]). Accessibility of the DNA was determined after formation of the hydrophobic cationic lipid– DNA complex, but prior to formation of a larger macromolecular structure. Dye binding was measured in an organic solvent system (A) and detergent (B). Data is expressed as Arbitrary Fluorescence Units and represent values obtained using an excitation wavelength of 509 nm and an emission wavelength of 533 nm. When the dye is added to polylysine condensed DNA, no fluorescence is observed. In contrast, the lipid complex exists as a structure that can still bind the dye. In the absence of solvents or detergents the complex adopts a macromolecular structure that prevents dye binding (see Fig. 5).

induced decreases in the dye exclusion indices were dependent on lipid composition; however, decreases in dye exclusion do not necessarily correlate with serum stability or transfection efficiency. It is known that addition of salt triggers significant changes in lipoplex structure [51]. In addition to facilitating dissociation of bound lipids [33], addition of salt promotes lipoplex aggregation and increases in lipid mixing reactions indicative of membrane fusion and / or lipid exchange [59]. Interestingly, increases in dye binding observed in the presence of salt were less evident when 10% serum was added. It can be argued that serum proteins could bind the lipoplex, providing a protective effect against destabilization.

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Fig. 5. TO-PRO-1 dye binding after lipoplex destabilization with 150 mM NaCl (from Ref. [58]). Dye exclusion indices, a parameter that measures dye binding relative to that observed using polylysine condensed DNA, were greater than 95% regardless of the lipoplex lipid composition (DODAC:DOPE (line 1 and line 2) or DODAC:DOPC (line 3 and line 4)) or the method employed to prepare the lipoplex (pre-formed cationic liposome / DNA aggregates (line 2 and line 3) or hydrophobic cationic lipid–DNA complex intermediate (line 1 and line 4)). An exclusion index approaching 100% was observed for polylysine condensed DNA. The percentage of dye exclusion reflected by bound TO-PRO-1 fluorescence has been calculated by (IOGP 2 I / IOGP )*100, where I is the fluorescence intensity in the absence (I) and presence (IOGP ) of detergent. The curve is representative of three different experiments. Arrow represents the addition of 150 mM NaCl.

It is known, for example, that as little as 1% serum can eliminate lipid mixing / membrane fusion reactions between cationic liposomes, in the absence of DNA [60]. Perhaps the most relevant measure of DNA protection is that obtained in assays measuring DNA degradation in the presence of serum. This is an important characteristic since these lipid-based carrier systems must mediate delivery of intact plasmid expression vectors in vitro in the presence of serum as well as in vivo following parenteral administration. Stability in the presence of serum is often modeled in defined reaction mixtures containing DNase I. This assay system, however, is problematic for two reasons. Similar to the DNA intercalating dye-binding assay described above, results obtained under physiological conditions are often quite different then those obtained in the absence of salt or

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serum. The second reason is related to the kinetics of the degradation reactions. These assays are typically completed using an experimental time frame and enzyme (serum) concentration that result in degradation of free plasmid, demonstrating that DNA associated in lipoplex formulations is better protected than free plasmid. As shown in Fig. 6, a proportion of DNA within lipoplexes remained intact in the presence of serum under conditions that resulted in complete degradation of the free plasmid expression vector. However, there was significant degradation and, if the assay time or DNase I concentration was increased, complete DNA hydrolysis is eventually observed. This is an important point: lipoplex technology is able to protect plasmid expression vectors for only a limited time period. In consideration of the previous point, it is worthwhile to describe what we hope to achieve, in terms of DNA protection, through use of lipoplex technology. Although gene therapy applications have been developed for ex vivo [61,62] and regionally administered lipoplex-formulated plasmid expression vectors (see next section), our interests are founded on development of lipid-based carrier formulations that exhibit activity following intravenous administration. A model of the delivery process following i.v. injection is shown in Fig. 7, a model advanced for lipid-based carriers of conventional small molecules used for the treatment of cancer. Analogous to liposomal drug formulations targeting cells outside the vascular compartment following i.v. administration, the lipoplex formulation must be stable within the blood compartment for a time period that is sufficient to allow regional localization in a disease site. Escape from the blood compartment may be a consequence of interactions with the cells (e.g., endothelial cells or egressing white blood cells) or passage through gaps between endothelial cells. Following extravasation into a tumor a number of events will determine the fate of regionally localized carriers. As indicated in Section 2, release of the plasmid expression vector from the lipid-based carrier in the interstitial space will result in plasmid degradation. The lipoplex must, therefore, remain stable until internalized by cells within the region of extravasation. Cell binding and internalization, by tumor-associated macrophages (TAMs), other host

Fig. 6. Serum stability assessment of DNA integrity (from Ref. [54]). M is the molecular weight marker. As controls, the plasmid expression vector (pINEXCatV2.0) was incubated in the absence and presence of serum followed by extraction and gel electrophoresis (second and third lanes, respectively). Lipid-based formulations prepared using the hydrophobic lipid–DNA complex intermediate (LDP) were prepared with the cationic lipid DODAC and various helper lipids at charge ratios (1 / 2) of 2:1 and lipid molar ratios of 1:1 (see Fig. 3). Stability of DNA in a cationic liposome (DODAC:DOPE) / DNA complex was also examined (lane 4). All lipoplex formulations were incubated in normal mouse serum for 1 h at 378C. The DNA was extracted using phenol–chloroform and the integrity of the DNA was analyzed by agarose gel electrophoresis.

derived cell populations and / or defined target cell populations, will be dependent on non-specific binding characteristics. Alternatively, surface-associated targeting molecules can be used to achieve redistri-

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Fig. 7. Steps required for delivery of lipid-based carriers to cells outside the vascular compartment following i.v. administration of liposomes. The macromolecular carrier formulations within the blood compartment (A) must escape. This may be a consequence of interactions with vascular endothelium (B) or white cells egressing into a disease site (C). The preferred mechanism of carrier extravasation will involve passage through gaps between endothelial cells (D), however this event is dependent on having formulations that are retained in the circulation for some time. Following extravasation into an extravascular site a number of events will determine the efficiency of carrier-mediated delivery of an associated biologically active agent. Release of the biologically active agent from the lipid-based delivery system in the interstitial space (E) can result in therapeutic benefits, however if the associated bioactive agent is DNA then there will be concerns about degradation. The carrier system can be internalized by host cells within the region (e.g. tumor-associated macrophages) (F). Direct interaction with a target cell population (G) in the extravascular site will be dependent on binding to the cell as well as internalization of the carrier formulation with its associated biologically active agent.

bution of the regionally localized lipoplexes such that target cell specific delivery is enhanced. Based on this model we propose that the ideal lipoplex-formulated plasmid expression vector must exhibit different physical and chemical properties depending on where the carriers are localized. Differences in localization are, of course, time dependent. Attributes of the lipoplex must favor plasmid stability in the blood compartment and following extravasation the attributes must change to enhance cell binding and internalization. As indicated in the subsequent sections, following internalization the

attributes will have to change again to support plasmid release.

6. The second barrier: binding to the cell membrane In general, lipoplex formulations that give rise to optimal transfection in vitro and in vivo are prepared such that the structure generated exhibits a net positive charge [63,64]. It has been argued that the positive charge facilitates binding to cell membranes,

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which have an anionic surface. We believe that this argument is too simplistic. As suggested above, lipoplex formulations tend to aggregate in the presence of salts and this aggregation reaction can be attributed to the surface charge of the carrier. A by-product of the aggregation reaction is a tendency for large particles to sediment, thereby facilitating cell contact, particularly in cultures of adherent cells. Studies have shown in cell culture that large systems are more effective transfection agents than small systems [65,66], although these studies typically evaluate liposome size prior to addition of DNA. Further, in our experience, dilution of lipoplex formulations into media causes significant aggregation, regardless of whether the starting lipoplexes were 300 nm or greater than 2000 nm [50,51]. For this reason it can be argued that the influence of lipoplex size on transfection has yet to be determined. This is important to the development of lipoplex technology, since size will play a critical role in controlling pharmacokinetic / biodistribution behavior, cell interactions and cell internalization pathways. More importantly, it is evident that mechanisms controlling lipoplex binding and delivery to cells in culture will not be applicable in vivo. This statement is supported by data demonstrating that optimal in vivo gene transfer requires a lower cationic lipid / DNA charge ratio than that used in cell culture [67–69]. The conclusions reached through in vitro studies, therefore, should be evaluated in context. Attributes that are optimal for cell culture transfection are not likely to be the same for lipoplexes designed for in vivo applications because the factors that impact formulation attributes are different. Furthermore, lipoplex attributes that support cell binding in vivo will differ depending on the route of administration.

6.1. Regional administration Administering lipoplex-formulated plasmids into specific regions can circumvent problems encountered following intravenous administration. Intramuscular, intraperitoneal, subcutaneous, intrathecal, intratracheal or intratumoral injection provides a means to increase delivery to target cells within these sites. Regional delivery of lipoplex-formulated plas-

mid expression vectors can promote transgene expression in specific tissues in vivo [68–71]. In particular, lipoplex formulations have been used to deliver DNA to lungs of animals after intratracheal administration [68] and successful expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the lung of animals led to clinical trials using lipid-based carriers of plasmid expression vectors. Another example of successful regional administration to direct the transfection of specific cells is intratumoral injection of lipoplexes for the treatment of melanoma [71]. The approach developed was based on generation of a systemic antitumor response following transfer of a major histocompatibility gene, HLA-B7, into tumor cells [72,73]. Pre-clinical results using this approach have demonstrated significant antitumor properties as well as immunity towards tumor cell inoculation [74]. Although regional approaches exhibit considerable potential, there are problems in evaluating lipoplex binding and delivery under these conditions. Similar to the intravenous route of administration, regionally administered lipoplexes will interact with soluble proteins, tissue elements (matrix proteins) and cells that will significantly change the attributes of the formulation and it is unclear how these interactions will affect cell binding or internalization.

6.2. Designing lipoplexes for intravenous applications Based on experience with conventional liposomal drug carriers, and in accordance to the model shown in Fig. 7, certain attributes are known to decrease the likelihood of cell binding in an extravascular site. Larger, aggregate structures ( . 200 nm) are eliminated rapidly following intravenous administration, mainly by uptake into the reticuloendothelial system and by lodging in small capillary beds (e.g., in the lung, liver and spleen) in comparison to small ( , 200 nm) structures [75]. Those structures that exhibit a positive or negative surface charge are eliminated more rapidly in comparison to neutral systems [76]. For lipoplex formulations exhibiting a cationic surface charge, anionic serum protein binding will certainly result in alterations of the surface characteristics. Protein binding is often associated with increases in plasma elimination rates, increased

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non-specific cell binding, increased phagocytic cell uptake, perturbations of the membrane structure and complement activation, all of which compromise the utility of systemically administered lipid-based drug carriers. Lipoplex formulations which aggregate under physiological conditions and exhibit a net charge (whether positive or negative) will have reduced access to cells outside the blood compartment simply as a consequence of mechanisms that enhance lipoplex elimination. Elimination is often restricted to select organs in the body, such as those with (i) microcapillary beds that can filter out macromolecular structures (e.g., lung), (ii) blood vessels that by their physical structure can allow passage of larger circulating macromolecules (e.g. fenestrated vessels in the liver or discontinuous vessels in the spleen) and / or (iii) cells capable of recognizing, binding and phagocytosis of foreign particulates (e.g., liver ¨ Kupffer cells, tumor-associated macrophages). It is not surprising, therefore, that significant levels of transgene expression are often observed in these organs following i.v. administration [77,78]. It is important to note that it is unclear which cells are transfected in these organs and that phagocytic cell uptake will likely result in lipoplex degradation. In order to redirect lipoplex formulations to cells in other sites it will be necessary to develop methods that result in small ( , 200 nm), neutral or chargeshielded structures. We believe that this can be achieved through use of surface grafted polyethylene glycol (PEG). As suggested in the previous section, significant advances in the use of lipoplex technology for plasmid delivery and gene therapy will require the development of macromolecular structures that contain features specific for stability in blood, controlled circulation lifetimes, disease site localization, and target cell specific binding and delivery. These lipoplex formulations will contain many different functional components in order that each of the desired attributes can be expressed optimally. Our approach to this multifunctional carrier is based on the premise that lipoplexes can be designed to transform their physical characteristics so that properties required during the delivery phase can be differentiated from those required for cell binding and internalization. In particular, it is known that PEG-modified lipids can be used to protect and

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stabilize lipid structures [79,80], including liposomes prepared with non-bilayer forming lipids [81,82] as well as emulsions of triacylglycerol [83]. Protection is facilitated by shielding of the membrane surface. This, in turn, reduces the rate of protein adsorption, inhibits aggregation reactions mediated between surfaces with multiple reactive groups [84], prevents (delays) interaction with cells [85] and effects significant increases in carrier circulation longevity [86]. Importantly, it is known that PEG lipids can also undergo spontaneous transfer between model lipid membranes [82,87]. For this reason the PEG lipids can be used as regulators of lipoplex surface properties and cell-binding attributes (Fig. 8). Studies with PEG-containing lipoplex formulations have recently been described by other researchers [88]. Initial studies from our laboratory suggest that lipoplex formulations prepared using the hydrophobic cationic lipid / DNA complex intermediate can be assembled with PEG lipids. As shown in Fig. 9, incorporation of PEG lipids into these lipoplex formulations resulted in a significant decrease in transfection activity, a decrease that may be correlated, at least in part, to decreases in cell binding (Fig. 9, insert). Loss of cell binding cannot, however, fully account for the significant decreases observed in transfection, therefore one must also suggest the possibility that PEG lipids may be interfering with other steps (e.g., internalization) involved in the transfection process. Regardless, it takes an optimist to consider such results exciting; however, it can be suggested that inhibition of non-specific cell association is an important development in the optimization of lipoplex technology. For these stabilized formulations, recovery of transfection activity will be dependent on loss / exchange of the PEG lipids as well as use of targeting ligands to facilitate cell specific binding.

6.3. Target specific binding The transfection efficiency of lipid-based plasmid delivery systems has been enhanced by the use of receptor-mediated targeting, which has emerged as a promising approach for the introduction of DNA into defined cell populations in vivo [89,90]. This approach involves targeting formulations to cells by covalently coupling targeting ligands to the carrier

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Fig. 8. The transformable liposome. Advances in the use of lipoplex technology will require the development of structures that contain features specific for stability to blood components, controlled circulation lifetimes, disease site localization and target cell specific delivery. Such liposomes must exhibit many different functional components such that each of the desired attributes can be expressed optimally. Our approach to this multifunctional carrier is based on the premise that liposomes can be designed to transform their physical characteristics so that properties required during the delivery phase of treatment can be differentiated from those required for a therapeutic effect. PEG lipids, which can undergo spontaneous transfer between membranes, can act as regulators of the lipoplex attributes.

system. The potential of targeting plasmid expression vectors through the use of carrier linked targeting molecules has been best demonstrated for the polymer polylysine. The preparation of targeting ligand– polylysine–DNA complexes has been described [91] and the asialoglycoprotein receptor (ASGPr) was one of the first receptors to be targeted via this approach [92]. ASGPr is expressed predominantly on hepatic cells and has been exploited for the internalization of DNA specifically to these target cells [92]. The transferrin receptor has also been used as a target to enhance binding to cells [93] and polylysine–transferrin–DNA complexes have been shown to be effective in delivering plasmid expression vectors to

various cell types over-expressing the transferrin receptor [94]. Increases in transfection efficiencies achieved through receptor-mediated DNA delivery can also be obtained for lipoplex technology. Targeting strategies typically involve the use of surface-associated targeting ligands. For example, folic acid-targeted lipoplex formulations have been successfully used to deliver plasmid DNA to tumor cells in vitro [95]. Alternatively, the methodology for attaching antibodies to liposomes for targeting purposes is well established [96] and these methods have been applied to enhance lipid-based plasmid delivery systems. In particular, lipoplex formulations tagged with

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Fig. 9. Incorporation of PEG lipids into lipoplex formulations can effectively inhibit transfection. Chloramphenicol acetyl transferase activity was measured following transfection of B16 / BL16 melanoma cells with lipoplex formulations (made using the hydrophobic complex intermediate) prepared using DODAC:DOPE and increasing percentages of a PEG (2000 MW) modified lipid (DSPE:PEG). Two micrograms DNA (pInexCAT) were added to each well and the cells were incubated in serum containing media for 4 h. Subsequently, the cells were washed and cultured for 48 h prior to measuring transgene expression. Insert: Lipoplex binding to cells was estimated using a flow cytometric assay. Murine B16 / BL16 melanoma cells were incubated with lipoplexes prepared with a fluorescent lipid marker (0.3% mol DiI) for 4 h at 378C. After removing these adherent cells by trypsin treatment, the cells were washed three times and analyzed by flow cytometry. The results were summarized by estimating the mean fluorescence intensity for each of the indicated lipoplex formulations. Values represent the mean of three different preparations6standard error of the mean.

monoclonal antibodies against Her2 / Neu have recently been developed to target plasmid expression in breast cancer cells in culture [97].

6.4. Cell binding, delivery and transfection efficiency: is there a correlation? Two of the most important barriers to transfection are association of the lipoplex formulation with the cell membrane and, subsequently, the entry of DNA into the cell. It is important, however, to recognize

that cell binding and intracellular delivery may not be sufficient to achieve transgene expression. To understand the mechanism(s) by which DNA enters the cell, we and others have developed simple assays to measure plasmid delivery to cells [63,98]. In order to differentiate between binding and internalization, the amount of plasmid associated with the cell can be measured at 37 and 48C [63] or in the presence and absence of metabolic inhibitors. Binding and uptake data generated in this manner can be used to address questions regarding the importance of cell binding

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and delivery in regulating transgene expression. For example, lipid composition is thought to be important in designing effective lipoplex formulations. As shown in Fig. 10, lipid composition mediated differences in transgene expression did not correlate to differences in the level of cell-associated DNA. These data suggest that lipid composition may affect the ability of the DNA to be expressed and this

Fig. 10. Influence of lipoplex lipid composition on transfection activity and DNA delivery (from Ref. [63]). Relative b-galactosidase activity was measured in murine B16 / BL6 melanoma cells 48 h following a 4 h transfection with plasmid expression vector which contained the gene for b-galactosidase (pCMVb). Lipoplex formulations of the plasmid expression vector were prepared using three different pre-formed liposomes such that the final liposomal lipid:DNA ratio was 10:1 (nmol lipid / mg DNA). Cells transfected using lipoplexes prepared with DODAC / DOPE liposomes complexed with pCMVb yielded the highest level of b-galactosidase activity (A), suggesting that this formulation was the most efficient in transfecting cells. Cell-associated DNA (B) was evaluated 4 h following transfection for the three different lipoplex formulations. The uptake of DNA in B16 cells (determined by subtracting cell-associated DNA levels measured at 48C from that measured at 378C) was not significantly different among the liposome formulations tested. These results suggest that although these formulations are effective at delivering DNA to the cells, the expression of the DNA is dependent on the lipid composition.

function may be independent of cell binding and cellular uptake.

7. The third barrier: DNA delivery and dissociation from the carrier Once a lipoplex interacts with a cell membrane, DNA enters the cell either directly through the plasma membrane or indirectly following endocytosis. Both entry routes require membrane destabilization and, regardless of whether the destabilized membrane is the plasma membrane or the endosomal membrane, these and / or other intracellular processes must also involve dissociation of the plasmid expression vector from the lipoplex. A pivotal manuscript addressing potential mechanism(s) of lipoplex-mediated membrane destabilization was authored by Xu and Szoka in 1996 [99]. These investigators developed a model which accounts for membrane destabilization reactions, required for DNA release into the cytoplasm, as well as reactions that lead to dissociation of DNA from the cationic lipids used for lipoplex preparation. We argue that the latter is likely one of the most important attributes, citing recent evidence from our laboratory that suggests that the transfection enhancing role of certain lipids is a consequence of specific interactions with cationic lipids used in lipoplex preparation, rather than roles involving membrane fusion (F. Wong, unpublished data). For successful transfection, it is also important that the internalized DNA is protected against enzymatic digestion. The stability of the DNA within the endosomal compartment becomes another important parameter, one that may be dependent on factors including the composition of the liposomal lipid, the cell type, as well as the method of lipoplex preparation. As indicated below, the lipid composition will play an important role in affecting the ability of the DNA to be processed following binding and entry into the cell. This may be affected by the strength of the ionic / hydrophobic interactions of the lipid with the DNA, influencing DNA stability as well as DNA release and delivery to the nucleus. Regardless, if endosomal escape is not achieved then the lipoplex, with its associated DNA, will eventually be degraded as it is transported from early to late

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endosomes, along the lysosomal degradation pathway [100]. It is currently thought that only a small proportion of DNA delivered via cationic liposomes escapes the lysosomal degradative pathway. It has yet to be established how many plasmids are required to reach the nucleus in order to achieve efficient transgene expression. Expression itself will be under the control of factors that are independent of nuclear delivery. Although protection against enzymatic degradation is important, additional interactions between cellular components and the internalized lipoplex must take place in order to dissociate the DNA from the lipid components. It has been demonstrated that lipoplex formulations injected directly into the nucleus are less able to promote transgene expression than plasmid DNA alone [101]. Once again we must contend with conflicting roles for the lipid-based carrier following internalization. The lipoplex formulation must protect the DNA against enzymatic degradation, but dissociation is required for gene expression. The problem of nuclear delivery is further confounded by the presence of nucleases in the cytoplasm and strategies will have to be developed to facilitate protection of the DNA as it is transferred into the nucleus.

7.1. Membrane destabilization reactions Although the mechanism of DNA transfer across the cell membrane is not well understood, it is generally believed that endocytosis is the major route of cellular uptake for lipoplex formulations [45,99,102]. This event, however, will be dependent on lipoplex size and lipid composition and the physical and chemical attributes of lipoplex formulations are often not well suited for cell uptake by endocytosis [103]. For this reason, mechanism(s) involving delivery through the cell’s plasma membrane must also be considered. Until recently, investigators have assigned a critical role in DNA delivery to lipoplex lipids that are known to promote fusion between membranes [51,59,60,99]. DOPE is a nonbilayer forming lipid that plays an important role in the regulation of membrane fusion [103] which until recently had been described as an essential lipoplex component. It has been proposed that lipoplex formulations

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can either fuse with the cell membrane or the endosomal membrane. According to the model proposed by Xu and Szoka, fusion can lead to escape of the DNA from the endosomal pathway into the cytosol. In addition, it is speculated that following endocytosis of the lipoplex, cationic lipids can induce flip-flop of anionic lipids from the cytoplasmic face of the endosomal membrane. Interactions with the anionic lipids result in destabilization of the complex and release of the nucleic acid. Evidence in support of this model has come from studies characterizing destabilization of lipoplex formulations following addition of anionic liposomes [99], a result that has been confirmed in our laboratory [58]. Destabilization reactions have been assessed by measuring: (i) lipid mixing between cationic lipid–DNA formulations and anionic liposomes, (ii) DNA intercalating dye binding (TOPRO-1) and (iii) increased sensitivity of DNA to DNase I mediated hydrolysis. The lipid mixing assay measures fluorescent lipid dilution in membranes as a consequence of lipid mixing, a process typically attributed to membrane fusion [104]. It has been established that the destabilization reaction observed following addition of negatively charged liposomes is a consequence of membrane interactions that bring about lipid mixing (Fig. 11). Our results have shown that lipid mixing reactions and lipoplex destabilization are enhanced by, but are not dependent on, the use of DOPE in the DNA formulation or the anionic membrane formulations used to induce destabilization. It is not clear, therefore, whether non-bilayer structures attributed to the presence of DOPE in the lipoplex are required for effective gene transfer. Other investigators have also failed to establish a link between lipid mixing reactions and transfection efficiency [105]. It is important to note that when employing preformed liposomes in the preparation of lipoplex formulations that these liposomes typically have a propensity to fuse. It has been established, for example, that addition of anionic liposomes to positively charged DOPE-containing liposomes, in absence of DNA, induces lipid mixing reactions and membrane fusion (refer to Fig. 2) [60]. Similarly, DNA addition to cationic liposomes causes lipid mixing reactions to occur between cationic liposomes [51]. In studies with pre-formed liposomes,

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Fig. 11. A model addressing potential mechanism(s) of lipoplex destabilization mediated by anionic membranes. Regardless of the method used to prepare the lipid-based DNA formulation, the residual cationic charge will facilitate binding to anionic membranes. Once close contact has been established between the two lipid structures the extent of membrane perturbations will be dependent on the lipid composition used. The net result will be to release bound lipids from the DNA. It was proposed by Xu and Szoka in 1996 [99] that membrane destabilization reactions are required for DNA release into the cytoplasm as well as reactions that lead to dissociation of DNA from the cationic lipids used for lipoplex preparation.

therefore, significant changes in the cationic liposomes occur after DNA addition and further destabilization of the complex induced by anionic liposomes may be reduced because of this. In contrast, such reactions cannot occur when using formulation technology where lipid-based complexes form following lipid (rather than liposome) binding to DNA [58]. Reduction in the transfection efficiency of certain lipoplex formulations may be due to a reduction in a capacity to undergo membrane interactions that foster lipid mixing. However, we and others have not been able to demonstrate this. In fact, results from our laboratory suggest that the ability of lipoplex formulations to exhibit membrane reactions, such as lipid mixing, decrease significantly as the formulations are mixed with salts and serum proteins. It can be suggested that the capacity of the lipoplexes to fuse with a membrane is reduced as a consequence of the initial DNA binding required to form a complex as well as salt and serum protein interactions. These interactions occur well before the lipop-

lex is endocytosed by a target cell. It is for this reason that we believe that the destabilization reactions that lead to DNA release from the lipid-based carrier are the most significant events in terms of defining transfection activity.

7.2. Dissociation of the lipid /DNA complex We have utilized the two-phase extraction system described in Section 4 to assess whether other lipids can destabilize the hydrophobic cationic lipid / DNA complex. This consists of forming cationic lipid / DNA complexes in an organic phase prior to addition of the other lipids to the organic phase [34]. The results demonstrated that anionic lipids (e.g., phosphatidylserine, phosphatidylglycerol and phosphatidylinositol) induced complex destabilization [34]. This is presumably a consequence of electrostatic interactions between the cationic and anionic lipids that compete with the binding reaction between cationic lipids and DNA. Such data is consistent with the model described by Xu and Szoka, where it was

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postulated that cationic lipids can induce a flip-flop of anionic lipids from the cytoplasmic face of the endosomal membrane resulting in destabilization of the complex and release of the nucleic acid [99]. It is of interest that studies have shown that anionic lipids may play a role in the dissociation of basic proteins (histones) from DNA [106]. We have shown that zwitterionic lipids containing the ethanolamine headgroup can destabilize the hydrophobic complex formed between cationic lipids and DNA. We have suggested that the ethanolamine headgroup facilitates dissociation of cationic lipids that are bound to DNA [54,58]. When rationalizing the beneficial properties of DOPE in comparison to DOPC, it is important to note that differences in the behavior of these lipids are often attributed to their hydration properties [107]. Lipids like DOPC adopt a lamellar phase as a consequence of both attractive (van der Waals) and repulsive (hydration) forces [108]. Similar forces play a role in the macromolecular structure adopted by DOPE, however an additional interaction between the amine group and the non-esterified oxygen of phosphate groups within and between lipid bilayers has been proposed [109]. Intra- and inter-molecular interactions and hydrationrepulsion forces help stabilize lipids into a bilayer (lamellar) phase. However, the added interaction between amine groups and phosphate groups in facing bilayers can promote local dehydration and facilitate close contact between membranes [110]. These events are also critical to the induction of membrane fusion. These effects, when combined with factors that influence the acyl chain packing properties (e.g., temperature, cholesterol addition), are thought to be responsible for the polymorphic phase behavior of hydrated lipid systems [111]. Although the potential for the amine group of PEs to interact with phosphate groups of phospholipids has been documented, a similar interaction could occur between the amine group of PEs and DNA phosphate groups. Such an interaction would serve to weaken the binding reaction between cationic lipids and DNA during the formation process [34,54,58]. This may be an important effect that ascribes an alternative role for phosphatidylethanolamine in lipidbased formulations used for DNA transfer, a role that involves release of cationic lipid-associated DNA rather than, or in addition to, membrane fusion. The

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importance of this destabilization reaction in governing factors that give rise to effective gene transfer needs to be evaluated.

7.3. Endosomal lysis and other modes of escape One simple mechanism of membrane disruption that has been proposed by Labat-Moleur et al. is a consequence of endosome swelling following endocytosis of lipopolyamine-formulated lipoplexes [112]. It is suggested that buffering due to endosomally localized lipopolyamine lipoplexes leads to osmotic swelling and subsequently membrane lysis. A similar approach has been proposed to enhanced transfection by addition of compounds (e.g., sucrose) to promote osmotic swelling or lysosomotropic drugs (e.g., chloroquine) during the transfection step [113]. Such approaches will only be applicable to in vitro transfection procedures unless strategies can be developed to co-deliver such agents with the lipoplex formulation. We would like to suggest another possibility, one that would be dependent on degradation of lipids used in the preparation of lipoplexes. Lipid hydrolysis can lead to the production of free fatty acids or lysolipids and endosome lysis may be attributed to the detergent-like properties of these lipids. Therefore, lipids effective in terms of mediating cytoplasmic delivery of DNA may have components (or properties) that have a propensity to lyse membranes. Several methods have been evaluated for their ability to augment the release of DNA from the endosomal compartment. One approach involves the use of pH-sensitive liposomes, which are formulated to destabilize under conditions of low pH [114]. Since delivery of DNA mediated by lipoplex formulations is thought to be via the endocytic pathway, pH-sensitive lipoplexes are believed to be particularly well suited for intracellular delivery of plasmid expression vectors. Wang and Huang [115] have demonstrated that pH-sensitive liposomes can be used to enhance membrane lysis at the lower pH of the endosome, effecting release of DNA to the cytoplasmic compartment. The problem with most approaches involving use of pH-sensitive liposomes concerns the fact that these systems are not stable in vivo. We believe that this specific problem can be

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overcome through use of PEG lipids, as described in Section 6.2. Another approach to enhance release from the endosomal compartment involves the use of lipoplex technology that incorporates peptides that can trigger endosome lysis or fusion [116]. These include proteins used by viruses to escape the endosomal compartment, such as hemagglutinin [117], as well as synthetic analogues designed to have membrane lytic activity [118]. Some of these viral proteins are well characterized and when reconstituted with lipids isolated from viral envelopes they can be used to form very efficient fusogenic delivery systems referred to as virosomes. In vivo studies using lipoplexes prepared using the viral fusion peptides have demonstrated effective DNA delivery into hepatocytes and renal artery following administration into the liver and kidney, respectively [93]. Some of the peptides used to augment cell transfection are known to form transmembrane channels [119], which can disrupt the plasma membrane as well as the endosome and release the DNA into the cytoplasm. In terms of in vivo applications we believe that benefits achieved in the transfection activity of lipoplex formulations prepared with fusion promoting and / or lytic peptides must be balanced against the potential of these systems to elicit immune responses.

is absent during cell division, it has been proposed that transfection would be more efficient in rapidly dividing cells and transgene expression would be dependent on the cell cycle. This has been observed in several studies characterizing plasmid delivery systems [120,121]. However, in vivo transfection can occur in muscle that consists of non-dividing cells (myoblasts) that maintain nuclear membrane integrity [122,123]. Studies with lipoplex formulations have also demonstrated that the efficiency of DNA transfer to the nucleus can be enhanced by incorporation of nuclear proteins in the lipoplex [124]. These nuclear proteins are unique in that they bear nuclear localization signals, which are thought to enhance trafficking of the DNA into the nucleus. There is little, if any, evidence supporting the contention that bound lipids play a role in facilitating transfer of the plasmid expression vectors into the nucleus. Studies on the interaction of polynucleotides and membranes, however, may help define such a role and it has been postulated that membrane / DNA interactions may provide another mechanism of controlling gene expression in cells, one based on dissociation of histones from DNA [106]. It is important to note that the association of nuclear localization signals does not necessarily protect the plasmid in the cytoplasm; other elements will have to be utilized in conjunction with nuclear localization signals to optimize nuclear delivery of intact plasmid expression vectors.

8. The fourth barrier: transfer into the nucleus Cationic lipid-mediated transfection is a relatively inefficient process, which is initiated by binding of the plasmid expression vector to the cell [64,98]. While a significant portion of the cell-associated DNA is internalized, only a small percentage of DNA is released from endosomes. Therefore, only a small amount of DNA will be available as free DNA in the cytoplasm, and this DNA will still be susceptible to degradation. This free DNA must still find its way to the nucleus. An unlikely alternative involves the complex itself entering the nucleus and subsequently dissociating in the nucleus in order for the DNA to be available for transcription. However, others have suggested that lipid dissociation occurs during transport into the nucleus. The nuclear membrane represents a critical barrier for effective transfection. Since the nuclear envelope

9. Summary – lipoplex design challenges Our interest in developing lipid-based carriers with attributes that change as a function of time following i.v. administration has been summarized here. Other investigators are focusing on developing model (reconstituted) viruses as efficient nucleic acid delivery systems. The three viral attributes that have attracted the greatest attention are (i) surface binding interactions [125], (ii) viral membrane fusion proteins [126] and (iii) nuclear targeting signals [127]. Based on what is known about viruses, there is general agreement that the development of more effective lipid-based DNA delivery systems will involve incorporating elements which control DNA condensation, utilize cell surface receptors while avoiding non-specific interactions, escape the endo-

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somal compartment and transfer the nucleic acid into the nucleus of the target cell. However, if effective lipoplex formulations must rely on incorporation of proteins to accomplish cell delivery, membrane fusion and DNA localization to the nucleus, then questions should be asked regarding the benefits of lipoplex technology. The primary advantages of lipoplex technology are that the methods used are simple, pharmaceutically viable and versatile. If the improvements being developed result in a complex technology that is difficult to develop for pharmaceutical use, then we should reconsider our approach. If the optimized lipoplex formulations prove to be as immunogenic as viral systems and improvements in transfection are a consequence of integration into the host genome, then we should again reconsider our approach. It is our belief that simple lipid-based DNA delivery systems can be refined based on principles already developed for liposomal drug delivery systems, although there are important differences. First, the plasmid delivery systems must promote intracellular delivery of the active agent. It is worth noting that advances in liposome technology are also focused on designing carriers that more effectively deliver associated bioactive agents into cells rather than in regions surrounding cells. The second important difference concerns the active agents used. Plasmid expression vectors have not been fully optimized for gene therapy applications. An important area of research not considered here involves restructuring the transgene to include strong promoters, enhancers, and other regulatory elements that will promote higher levels of the transcribed message and translated protein once the DNA gains access to the nucleus [128]. Improvements in lipoplex technology will likely arise as a consequence of using better designed lipid-based carriers to deliver optimized expression vectors. Our focus will remain on carefully designed and characterized lipid-based carrier systems.

Acknowledgements The Medical Research Council of Canada and Inex Pharmaceuticals Corp. supports research on gene transfer formulations in M. Bally’s laboratory.

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D. Reimer was a MRC Postdoctoral Fellow. E. Wasan holds a fellowship from the British Columbia Science Council. F. Wong is a recipient of a Cancer Research Society Studentship.

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