Biological barriers to cellular delivery of lipid-based DNA carriers
Identifieur interne : 002824 ( Istex/Corpus ); précédent : 002823; suivant : 002825Biological barriers to cellular delivery of lipid-based DNA carriers
Auteurs : Marcel B. Bally ; Pierrot Harvie ; Frances M. P Wong ; Spencer Kong ; Ellen K. Wasan ; Dorothy L. ReimerSource :
- Advanced Drug Delivery Reviews [ 0169-409X ] ; 1999.
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.
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DOI: 10.1016/S0169-409X(99)00034-4
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Biological barriers to cellular delivery of lipid-based DNA carriers</title><author><name sortKey="Bally, Marcel B" sort="Bally, Marcel B" uniqKey="Bally M" first="Marcel B" last="Bally">Marcel B. Bally</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: mbally@bccancer.bc.ca</mods:affiliation></affiliation></author><author><name sortKey="Harvie, Pierrot" sort="Harvie, Pierrot" uniqKey="Harvie P" first="Pierrot" last="Harvie">Pierrot Harvie</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Wong, Frances M P" sort="Wong, Frances M P" uniqKey="Wong F" first="Frances M. P" last="Wong">Frances M. P Wong</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Kong, Spencer" sort="Kong, Spencer" uniqKey="Kong S" first="Spencer" last="Kong">Spencer Kong</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Wasan, Ellen K" sort="Wasan, Ellen K" uniqKey="Wasan E" first="Ellen K" last="Wasan">Ellen K. Wasan</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Reimer, Dorothy L" sort="Reimer, Dorothy L" uniqKey="Reimer D" first="Dorothy L" last="Reimer">Dorothy L. Reimer</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1016/S0169-409X(99)00034-4</idno><idno type="url">https://api-v5.istex.fr/document/1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">002824</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002824</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Biological barriers to cellular delivery of lipid-based DNA carriers</title><author><name sortKey="Bally, Marcel B" sort="Bally, Marcel B" uniqKey="Bally M" first="Marcel B" last="Bally">Marcel B. Bally</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: mbally@bccancer.bc.ca</mods:affiliation></affiliation></author><author><name sortKey="Harvie, Pierrot" sort="Harvie, Pierrot" uniqKey="Harvie P" first="Pierrot" last="Harvie">Pierrot Harvie</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Wong, Frances M P" sort="Wong, Frances M P" uniqKey="Wong F" first="Frances M. P" last="Wong">Frances M. P Wong</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Kong, Spencer" sort="Kong, Spencer" uniqKey="Kong S" first="Spencer" last="Kong">Spencer Kong</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Wasan, Ellen K" sort="Wasan, Ellen K" uniqKey="Wasan E" first="Ellen K" last="Wasan">Ellen K. Wasan</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Reimer, Dorothy L" sort="Reimer, Dorothy L" uniqKey="Reimer D" first="Dorothy L" last="Reimer">Dorothy L. Reimer</name><affiliation><mods:affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Advanced Drug Delivery Reviews</title><title level="j" type="abbrev">ADR</title><idno type="ISSN">0169-409X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">38</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="291">291</biblScope><biblScope unit="page" to="315">315</biblScope></imprint><idno type="ISSN">0169-409X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0169-409X</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Marcel B Bally</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string><json:string>E-mail: mbally@bccancer.bc.ca</json:string></affiliations></json:item><json:item><name>Pierrot Harvie</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string></affiliations></json:item><json:item><name>Frances M.P Wong</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string></affiliations></json:item><json:item><name>Spencer Kong</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string></affiliations></json:item><json:item><name>Ellen K Wasan</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string></affiliations></json:item><json:item><name>Dorothy L Reimer</name><affiliations><json:string>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>DNA transfer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Transfection</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Cationic lipids</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Gene therapy</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><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.</abstract><qualityIndicators><score>8</score><pdfWordCount>13656</pdfWordCount><pdfCharCount>90763</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>25</pdfPageCount><pdfPageSize>546 x 744 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>276</abstractWordCount><abstractCharCount>1897</abstractCharCount><keywordCount>4</keywordCount></qualityIndicators><title>Biological barriers to cellular delivery of lipid-based DNA carriers</title><pii><json:string>S0169-409X(99)00034-4</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Advanced Drug Delivery Reviews</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0169-409X</json:string></issn><pii><json:string>S0169-409X(00)X0048-8</json:string></pii><volume>38</volume><issue>3</issue><pages><first>291</first><last>315</last></pages><genre><json:string>journal</json:string></genre></host><categories><wos><json:string>science</json:string><json:string>pharmacology & pharmacy</json:string></wos><scienceMetrix><json:string>health sciences</json:string><json:string>clinical medicine</json:string><json:string>pharmacology & pharmacy</json:string></scienceMetrix><inist><json:string>sciences appliquees, technologies et medecines</json:string><json:string>sciences biologiques et medicales</json:string><json:string>sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1016/S0169-409X(99)00034-4</json:string></doi><id>1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3</id><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api-v5.istex.fr/document/1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api-v5.istex.fr/document/1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api-v5.istex.fr/document/1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">Biological barriers to cellular delivery of lipid-based DNA carriers</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1999 Elsevier Science B.V.</p></availability><date>1999</date></publicationStmt><notesStmt><note type="content">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.</note><note type="content">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].</note><note type="content">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,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and various helper lipids such that the cationic lipid to DNA phosphate ratio (+/−) 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 mean±standard error of the mean.</note><note type="content">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).</note><note type="content">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−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.</note><note type="content">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 (+/−) 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 37°C. The DNA was extracted using phenol–chloroform and the integrity of the DNA was analyzed by agarose gel electrophoresis.</note><note type="content">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.</note><note type="content">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.</note><note type="content">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 37°C. 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 preparations±standard error of the mean.</note><note type="content">Fig. 10: Influence of lipoplex lipid composition on transfection activity and DNA delivery (from Ref. [63]). Relative β-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 β-galactosidase (pCMVβ). 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/μg DNA). Cells transfected using lipoplexes prepared with DODAC/DOPE liposomes complexed with pCMVβ yielded the highest level of β-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 4°C from that measured at 37°C) 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.</note><note type="content">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.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Biological barriers to cellular delivery of lipid-based DNA carriers</title><author xml:id="author-0000"><persName><forename type="first">Marcel B</forename><surname>Bally</surname></persName><email>mbally@bccancer.bc.ca</email><note type="correspondence"><p>Corresponding author. Tel.: +1-604-877-6098, extn. 3191; fax: +1-604-877-6011</p></note><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Pierrot</forename><surname>Harvie</surname></persName><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Frances M.P</forename><surname>Wong</surname></persName><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Spencer</forename><surname>Kong</surname></persName><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Ellen K</forename><surname>Wasan</surname></persName><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Dorothy L</forename><surname>Reimer</surname></persName><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation></author><idno type="istex">1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3</idno><idno type="DOI">10.1016/S0169-409X(99)00034-4</idno><idno type="PII">S0169-409X(99)00034-4</idno></analytic><monogr><title level="j">Advanced Drug Delivery Reviews</title><title level="j" type="abbrev">ADR</title><idno type="pISSN">0169-409X</idno><idno type="PII">S0169-409X(00)X0048-8</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">38</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="291">291</biblScope><biblScope unit="page" to="315">315</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>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.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>DNA transfer</term></item><item><term>Transfection</term></item><item><term>Cationic lipids</term></item><item><term>Gene therapy</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api-v5.istex.fr/document/1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="gr8"></istex:entity><istex:entity SYSTEM="gr9" NDATA="IMAGE" name="gr9"></istex:entity><istex:entity SYSTEM="gr10" NDATA="IMAGE" name="gr10"></istex:entity><istex:entity SYSTEM="gr11" NDATA="IMAGE" name="gr11"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>ADR</jid><aid>781</aid><ce:pii>S0169-409X(99)00034-4</ce:pii><ce:doi>10.1016/S0169-409X(99)00034-4</ce:doi><ce:copyright type="full-transfer" year="1999">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>Biological barriers to cellular delivery of lipid-based DNA carriers</ce:title><ce:author-group><ce:author><ce:given-name>Marcel B</ce:given-name><ce:surname>Bally</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>mbally@bccancer.bc.ca</ce:e-address></ce:author><ce:author><ce:given-name>Pierrot</ce:given-name><ce:surname>Harvie</ce:surname></ce:author><ce:author><ce:given-name>Frances M.P</ce:given-name><ce:surname>Wong</ce:surname></ce:author><ce:author><ce:given-name>Spencer</ce:given-name><ce:surname>Kong</ce:surname></ce:author><ce:author><ce:given-name>Ellen K</ce:given-name><ce:surname>Wasan</ce:surname></ce:author><ce:author><ce:given-name>Dorothy L</ce:given-name><ce:surname>Reimer</ce:surname></ce:author><ce:affiliation><ce:textfn>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +1-604-877-6098, extn. 3191; fax: +1-604-877-6011</ce:text></ce:correspondence></ce:author-group><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>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.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>DNA transfer</ce:text></ce:keyword><ce:keyword><ce:text>Transfection</ce:text></ce:keyword><ce:keyword><ce:text>Cationic lipids</ce:text></ce:keyword><ce:keyword><ce:text>Gene therapy</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Biological barriers to cellular delivery of lipid-based DNA carriers</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Biological barriers to cellular delivery of lipid-based DNA carriers</title></titleInfo><name type="personal"><namePart type="given">Marcel B</namePart><namePart type="family">Bally</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><affiliation>E-mail: mbally@bccancer.bc.ca</affiliation><description>Corresponding author. Tel.: +1-604-877-6098, extn. 3191; fax: +1-604-877-6011</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Pierrot</namePart><namePart type="family">Harvie</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Frances M.P</namePart><namePart type="family">Wong</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Spencer</namePart><namePart type="family">Kong</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Ellen K</namePart><namePart type="family">Wasan</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Dorothy L</namePart><namePart type="family">Reimer</namePart><affiliation>Medical Oncology–Advanced Therapeutics, British Columbia Cancer Agency and Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">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.</abstract><note type="content">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.</note><note type="content">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].</note><note type="content">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,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and various helper lipids such that the cationic lipid to DNA phosphate ratio (+/−) 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 mean±standard error of the mean.</note><note type="content">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).</note><note type="content">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−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.</note><note type="content">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 (+/−) 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 37°C. The DNA was extracted using phenol–chloroform and the integrity of the DNA was analyzed by agarose gel electrophoresis.</note><note type="content">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.</note><note type="content">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.</note><note type="content">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 37°C. 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 preparations±standard error of the mean.</note><note type="content">Fig. 10: Influence of lipoplex lipid composition on transfection activity and DNA delivery (from Ref. [63]). Relative β-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 β-galactosidase (pCMVβ). 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/μg DNA). Cells transfected using lipoplexes prepared with DODAC/DOPE liposomes complexed with pCMVβ yielded the highest level of β-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 4°C from that measured at 37°C) 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.</note><note type="content">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.</note><subject><genre>Keywords</genre><topic>DNA transfer</topic><topic>Transfection</topic><topic>Cationic lipids</topic><topic>Gene therapy</topic></subject><relatedItem type="host"><titleInfo><title>Advanced Drug Delivery Reviews</title></titleInfo><titleInfo type="abbreviated"><title>ADR</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">19990820</dateIssued></originInfo><identifier type="ISSN">0169-409X</identifier><identifier type="PII">S0169-409X(00)X0048-8</identifier><part><date>19990820</date><detail type="issue"><title>Membrane Destabilization for Improved Cytosolic Delivery</title></detail><detail type="volume"><number>38</number><caption>vol.</caption></detail><detail type="issue"><number>3</number><caption>no.</caption></detail><extent unit="issue pages"><start>195</start><end>338</end></extent><extent unit="pages"><start>291</start><end>315</end></extent></part></relatedItem><identifier type="istex">1136B39EF8FA70CF2F22C6DDBE2770F8C3662FE3</identifier><identifier type="DOI">10.1016/S0169-409X(99)00034-4</identifier><identifier type="PII">S0169-409X(99)00034-4</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Elsevier Science B.V., ©1999</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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