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• W2163537252 abstract "The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40–60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitromodel of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to ∼35%. Treatment with 1 µm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to ∼75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5–10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector. The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40–60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitromodel of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to ∼35%. Treatment with 1 µm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to ∼75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5–10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector. antisense oligonucleotide (ON) phosphodiester antisense oligonucleotides in anionic liposomes phosphorothioate antisense oligonucleotides in anionic liposomes 6-cyano-7-nitroquinoxaline-2,3-dione oligonucleotides labeled with Cy3 dimethylaminoethane carbamoyl cholesterol dioleoyl phosphatidic acid dioleoyl phosphatidylcholine dioleoyl phosphatidylethanolamine dioleoyl phosphatidylglycerol dioleoyl phosphatidylserine dioleoyl trimethylammoniumpropane dizocilpine pifithrin-α sScr, and sMm, phosphorothioate antisense, scrambled, and mismatch oligonucleotides respectively ammonium ferrothiocyanate enhanced green fluorescent protein Madin-Darby canine kidney cells Chinese hamster ovary Selective inhibition of gene expression with antisense oligonucleotides (AsONs)1 is both a popular technique for probing fundamental questions of neuroscience (1Sattler R. Xiong Z. Lu W.Y. Hafner M. MacDonald J.F. Tymianski M. Science. 1999; 284: 1845-1848Crossref PubMed Scopus (708) Google Scholar) and a potential therapeutic strategy for the treatment of neurodegenerative diseases (2Gonzalez-Zulueta M. Ensz L.M. Mukhina G. Lebovitz R.M. Zwacka R.M. Engelhardt J.F. Oberley L.W. Dawson V.L. Dawson T.M. J. Neurosci. 1998; 18: 2040-2055Crossref PubMed Google Scholar). However, the elegance of the antisense concept belies the considerable challenge of their intracellular delivery (3Bally M.B. Harvie P. Wong F.M. Kong S. Wasan E.K. Reimer D.L. Adv. Drug Deliv. Rev. 1999; 38: 291-315Crossref PubMed Scopus (166) Google Scholar). Chemical modifications of ONs that enhance nuclease-resistance (e.g. phosphorothioates) have poor cellular uptake (∼5–10%) and cause non-sequence-specific effects, raising questions about the efficacy and selectivity of antisense drugs (4Stein C.A. Nat. Biotechnol. 1999; 17: 209Crossref PubMed Scopus (65) Google Scholar). Cationic lipids and polycationic polymers used as ON delivery vectors have met with limited success due to a number of variables that seem to affect vector performance (3Bally M.B. Harvie P. Wong F.M. Kong S. Wasan E.K. Reimer D.L. Adv. Drug Deliv. Rev. 1999; 38: 291-315Crossref PubMed Scopus (166) Google Scholar, 5Zabner J. Fasbender A.J. Moninger T. Poellinger K.A. Welsh M.J. J. Biol. Chem. 1995; 270: 18997-19007Abstract Full Text Full Text PDF PubMed Scopus (1293) Google Scholar). Mechanistic aspects of cationic lipid-mediated delivery are poorly understood because of the physical heterogeneity of cationic lipid-ON complexes (6Jaaskelainen I. Monkkonen J. Urtti A. Biochim. Biophys. Acta. 1994; 1195: 115-123Crossref PubMed Scopus (101) Google Scholar) that may contribute to their toxicity toward several cell types (7Hartmann G. Krug A. Bidlingmaier M. Hacker U. Eigler A. Albrecht R. Strasburger C.J. Endres S. J. Pharmacol. Exp. Ther. 1998; 285: 920-928PubMed Google Scholar). Application of antisense technology to the nervous system presents an even greater challenge because of the post-mitotic nature of neurons and their exquisite sensitivity to their microenvironment. Cationic lipids and polymers have been used to deliver nucleic acids to neurons, generally at efficiencies of 0.5–5% (8Kaech S. Kim J.B. Cariola M. Ralston E. Brain Res. Mol. Brain Res. 1996; 35: 344-348Crossref PubMed Scopus (41) Google Scholar). Factors that influence transgene expression or target protein inhibition include neuronal maturity at the time of transfection, the type of cationic lipid used (8Kaech S. Kim J.B. Cariola M. Ralston E. Brain Res. Mol. Brain Res. 1996; 35: 344-348Crossref PubMed Scopus (41) Google Scholar), and the net charge of the lipid-DNA complex (9Schwartz B. Benoist C. Abdallah B. Scherman D. Behr J.P. Demeneix B.A. Hum. Gene Ther. 1995; 6: 1515-1524Crossref PubMed Scopus (81) Google Scholar). Cationic lipidsper se have also been reported to be toxic to neurons (8Kaech S. Kim J.B. Cariola M. Ralston E. Brain Res. Mol. Brain Res. 1996; 35: 344-348Crossref PubMed Scopus (41) Google Scholar,10Azzazy H.M.E. Hong K. Wu M.-C. Gross G.W. Brain Res. 1995; 695: 231-236Crossref PubMed Scopus (6) Google Scholar). Glutamate, the main excitatory neurotransmitter in the brain, plays a central role in the pathogenesis of stroke, epilepsy, and neurodegenerative diseases such as Alzheimer's disease. Excitotoxicity results in increased translation (11Chen R.W. Chuang D.M. J. Biol. Chem. 1999; 274: 6039-6042Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar) and stabilization (12Jimenez G.S. Khan S.H. Stommel J.M. Wahl G.M. Oncogene. 1999; 18: 7656-7665Crossref PubMed Scopus (167) Google Scholar) of the p53 protein, which in turn alters the levels of redox proteins (13Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2218) Google Scholar), resulting in neuronal loss. p53 has also been shown to accumulate in mitochondria, leading to mitochondrial dysfunction and activation of the caspase cascade (14Marchenko N.D. Zaika A. Moll U.M. J. Biol. Chem. 2000; 275: 16202-16212Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). As further proof of the involvement of p53 in neurodegeneration, adenovirus-mediated overexpression of p53 causes apoptosis in cultured hippocampal neurons (15Jordan J. Galindo M.F. Prehn J.H. Weichselbaum R.R. Beckett M. Ghadge G.D. Roos R.P. Leiden J.M. Miller R.J. J. Neurosci. 1997; 17: 1397-1405Crossref PubMed Google Scholar), whereas neurons from p53 null mice are resistant to glutamate (16Xiang H. Hochman D.W. Saya H. Fujiwara T. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1996; 16: 6753-6765Crossref PubMed Google Scholar), DNA-damaging agents, and hypoxia (17Morrison R.S. Kinoshita Y. Cell Death Differ. 2000; 7: 868-879Crossref PubMed Scopus (125) Google Scholar). Suppression of p53 expression by AsONs protects neurons from apoptosis induced by DNA damage (18Chen R.W. Saunders P.A. Wei H. Li Z. Seth P. Chuang D.M. J. Neurosci. 1999; 19: 9654-9662Crossref PubMed Google Scholar). Although antisense-mediated inhibition of p53 protein expression has therapeutic potential in conditions where neuronal survival is compromised, precise delivery of the oligonucleotides to neurons is imperative for this potential to be fully realized. As the requirement for a net positive charge on transfection-competent cationic lipid-DNA complexes has been questioned by several recent reports (9Schwartz B. Benoist C. Abdallah B. Scherman D. Behr J.P. Demeneix B.A. Hum. Gene Ther. 1995; 6: 1515-1524Crossref PubMed Scopus (81) Google Scholar, 19Xu Y. Hui S.W. Frederik P. Szoka Jr., F.C. Biophys. J. 1999; 77: 341-353Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 20Son K.K. Patel D.H. Tkach D. Park A. Biochim. Biophys. Acta. 2000; 1466: 11-15Crossref PubMed Scopus (57) Google Scholar, 21Wangerek L.A. Dahl H.-H.M. Senden T.J. Carlin J.B. Jans D.A. Dustan D.E. Ioannou P.A. Williamson R. Forrest S.M. J. Gene Med. 2001; 3: 72-81Crossref PubMed Scopus (25) Google Scholar), we explored the ability of anionic lipids for DNA delivery to neurons. In the present study, zwitterionic and anionic phospholipids were used to design liposomal vectors (anionic liposomes) for oligonucleotide encapsulation. Antisense ONs to p53 delivered by anionic liposomes protected hippocampal neurons from glutamate-induced death by sequence-specific down-regulation of p53 without any discernible toxicity. Uptake of Cy3-labeled ONs delivered by anionic liposomes was studied in primary neurons, immortalized fibroblasts, and cell lines derived from the liver, kidney, ovary, and cervix. Anionic liposomes were successful in delivering Cy3ONs to the entire population of cells within 1 h, regardless of the cell type. The 18-mer p53 antisense ON used in this study targets the translation initiation site of the rat p53 mRNA and is complementary to nucleotides 21–38 (5′-CTGTGAATCCTCCATGAC-3′, GenBankTM accession numberX13058 (22Soussi T. Caron de Fromentel C. Breugnot C. May E. Nucleic Acids Res. 1988; 16: 11384Crossref PubMed Scopus (116) Google Scholar)) with 50% GC content for optimal hybridization. Scrambled (5′-TCGATCTACGACTGACTC-3′) and mismatch (5′-GAGTGAATGATCCATGGG-3′) sequences were also designed for use as negative controls. The sequences had no similarity to other mammalian genes (BLAST search (23Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar)) and exhibited minimal self-complementarity (Vector NTI, Informax, Inc.). All ONs, synthesized as lyophilized powders by Midland Certified Reagent Co. (Midlands, TX), were reconstituted in sterile, nuclease-free Tris-EDTA buffer (10 mm Tris HCl, 1 mm EDTA, (pH 7.4)) and stored at −20 °C. The concentrations of ONs in solution were routinely determined by absorbance measurements at 260 nm. Cy3-labeled oligonucleotides were synthesized by Integrated DNA Technologies, Coralville, IA. DOPC, DOPG, DOPS, DOPA, DOPE, DC-Chol, and DOTAP were purchased from Avanti Polar Lipids, Alabaster, AL and stored at −20 °C as stock solutions of 2 mg/ml in chloroform. Anionic liposomes were prepared by a modification of the classic film hydration-extrusion procedure. Briefly, the lipid mixture was dried to a thin film under a stream of high purity nitrogen and hydrated with a solution of ONs in 10 mm HEPES buffer (pH 7.4) with 5 mm NaCl (except when indicated otherwise) with intermittent heating and vortexing. After complete hydration, the suspension was transferred to a Liposofast™ miniextruder system (Avestin, Inc., Ottawa, Canada) and extruded through a series of polycarbonate membranes down to a pore size of 0.2 µm. Unencapsulated ONs were removed by loading the liposomes on a Sephadex G-50 column (7 × 0.5 cm, preequilibrated in hydration buffer) and centrifuging for 2 min at 180 × g. Liposomes were eluted in the void volume, and unencapsulated ONs were eluted in subsequent fractions. Purified liposomes were stored at 4 °C until use. Cationic DC-Chol/DOPE (1/1 molar ratio) liposomes were prepared in 10 mm HEPES buffer and extruded to 200 nm. The liposomes were diluted in 5% w/v glucose, complexed with ONs in various charge ratios, and used immediately after complex formation. Commercial cationic liposomal transfection reagents TransFastTM and Tfx-20™ were obtained from Promega(Madison, WI) and used according to the manufacturer's instructions. Size analysis of liposomes was performed by quasi-elastic laser light scattering using a Nicomp Model 370 submicron particle sizer (Particle Sizing Systems, Santa Barbara, CA). At least one million particles were analyzed for each formulation, and Gaussian or Nicomp distributions were chosen based on the χ2 goodness of fit (24Winterhalter M. Lasic D.D. Chem. Phys. Lipids. 1993; 64: 35-43Crossref PubMed Scopus (77) Google Scholar). Aliquots (∼ 20 µl) of the liposome suspensions were diluted to 500 µl with distilled water, and 500 µl of chloroform/methanol (1:1 v/v) was added to dissolve the liposomes. Aqueous and organic phases (containing the ONs and lipids, respectively) were separated by centrifugation at 1400 ×g for 10 min. This extraction procedure was repeated twice, and organic solvents dissolved in the aqueous phase were removed by heating in a 95 °C water bath for 15 min. Known volumes of the extracted ONs were diluted to 100 µl with Tris-EDTA buffer (10 mm Tris HCl, 1 mm EDTA) and loaded onto a 96-well plate. An equal volume of a 1:200 dilution of OliGreenTM (Molecular Probes, Eugene, OR) was added to the wells. The fluorescence increase upon binding of the dye to ON was measured using a FLUOStar microplate fluorometer (BMG Labtechnologies GmbH, Offenburg, Germany) with excitation and emission wavelengths of 480 and 535 nm. Because OliGreen™ exhibits significant base selectivity, the amount of ON in the liposomes was calculated from standard curves generated with a known concentration of that particular ON in solution. For Cy3-labeled ONs, Cy3 fluorescence in the aqueous phase after extraction was measured directly at excitation and emission wavelengths of 544 and 590 nm. The amount of ONs present in the extracted aqueous phase relative to the amount initially added to the lipid film was used to calculate the percent ON encapsulated in the liposomes. Loss of phospholipid during liposome preparation was determined by adding chloroform and ammonium ferrothiocyanate (AFT) to the dried extracted organic phases. The mixture was vortexed to induce formation of the colored AFT-phospholipid complex that partitions into the chloroform phase (25Stewart J.C. Anal. Biochem. 1980; 104: 10-14Crossref PubMed Scopus (1510) Google Scholar), and absorbance of the complex was measured at 475 nm (Beckman Instruments, Irvine, CA). Primary cultures of hippocampal neurons were prepared from neonatal rat pups (P1 or P2) as previously described (26Dubinsky J.M. J. Neurosci. 1993; 13: 623-631Crossref PubMed Google Scholar). Neurons were plated at a density of 60,000 cells/cm2 onto polylysine-coated plastic 12-well plates for the toxicity experiments or 100-mm dishes (Becton Dickinson, Franklin Lakes, NJ) for the immunoprecipitation studies in neurobasal medium with B27 supplements (Life Technologies, Inc.) and 0.5 mmglutamine. Fluorodeoxyuridine (15 µg/ml) was added to the cultures 24 h after plating to inhibit glial growth. Under these culture conditions, the survival and growth of non-neuronal cells was minimized. Cells were maintained at 37 °C in 95% air, 5% CO2 and were used between 6–8 days in vitro. ONs (unencapsulated or in liposomes) were added to the culture medium for 3 h at final concentrations of 0.1 to 5 µm, depending upon the experimental paradigm, and the neurons were then exposed to 50 µm glutamate. MK-801 and CNQX (final concentrations 20 µm each) were added 1–2 min before glutamate addition, and pifithrin-α (final concentration 10 µm) was added 3 h before glutamate addition. Neuronal survival was assessed by an observer blinded to the treatments 48 h after glutamate exposure by counting viable cells in preselected fields based on trypan blue exclusion (27Dubinsky J.M. Kristal B.S. Elizondo-Fournier M. J. Neurosci. 1995; 15: 7071-7078Crossref PubMed Google Scholar). The ratio of viable cells to the total number of neurons in the preselected fields was calculated for quantifying survival. Neurons (∼ 5 million cells/100-mm dish) were treated with 1 µm p53 antisense or scrambled ONs in anionic liposomes for 3 h and exposed to glutamate for 15 h. Cells were detached by scraping and sonicated in lysis buffer containing 0.1% SDS, 0.1% glycerol in 85 mm Tris HCl (pH 6.8) and protease inhibitor mixture set III (Calbiochem). After preclearing with Protein G-agarose (ImmunoPure®, Pierce), lysates were immunoprecipitated with the G59-12 monoclonal p53 antibody (2 µg/million cells, Pharmingen, San Diego, CA) and protein G-agarose. Immunoprecipitates and p53 positive control (Oncogene Research Products, Cambridge, MA) were resolved by 15% SDS-polyacrylamide gel electrophoresis, and proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Blots were incubated with the CM1 rabbit polyclonal p53 antibody (1:1000, Novocastra Laboratories, Newcastle upon Tyne, UK) and then probed with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:5000, Chemicon International, Inc., Temecula, CA). Detection was performed by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech), and p53 levels were quantified using a Personal Densitometer SI and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Neurons were cultured on 8-chambered glass slides (Nalgene Nunc, Napaville, IL) in minimum essential medium with 10% NuSerum (Collaborative Research). A plasmid coding for the enhanced green fluorescent protein driven by a cytomegalovirus promoter (pEGFP-N1, CLONTECH, Palo Alto, CA) was condensed with polyethyleneimine (Aldrich) and encapsulated in anionic DOPC/DOPG liposomes as described above. Neurons were incubated with pEGFP-N1 either alone or in anionic liposomes for 48 h in serum-containing medium, after which they were fixed in 4% paraformaldehyde and imaged for EGFP expression. The Chinese hamster ovary cell line CHO-K1, human hepatoma cell line HuH-7, cervical carcinoma cells HeLa, canine kidney cell line MDCK, and mouse embryonic fibroblast cell line, MEF-1 (ATCC, Manassas, VA), were cultured on eight-chambered glass slides in Dulbecco's modified Eagle's medium containing penicillin and streptomycin with 10% cosmic calf serum. Cy3-labeled oligonucleotides were added to the culture medium, either without any delivery vector, encapsulated in anionic DOPC/DOPG liposomes, or complexed to cationic DC-Chol/DOPE liposomes at a final concentration of 1 µm. After 1 h, the cells were rinsed with phosphate-buffered saline and fixed. Imaging was performed on a Leica TCS 4D confocal microscope (Deerfield, IL) equipped with an argon/krypton laser. The entire volume of the cells was scanned in 0.5-µm increments, and optimal images were obtained by averaging 16 images in the line-scan mode at the same fixed gains for all experiments. All fluorescence images presented in figures were equally contrast-enhanced using Adobe Photoshop (Adobe, Mountain View, CA). A minimum of 600 cells for each cell line and 50–100 neurons were visualized for each condition, and the presence or absence of intracellular Cy3 fluorescence (568-nm excitation, LP590-nm emission) or EGFP expression (488 nm excitation, LP515 nm emission) was noted. Data were analyzed by one-way or two-way analysis of variance with the Bonferroni post-test (GraphPad Prism®, GraphPad Software, Inc., San Diego, CA). Liposomes composed of DOPC and 12 mol % anionic lipids DOPG, DOPS, or DOPA were monodisperse suspensions with narrow Gaussian size distributions (Fig. 1A) and encapsulated 40–60% of the initial ON amount (TableI) depending on the liposome composition. The amount of ON encapsulated in the liposomes was measured using the OliGreenTM dye, which is highly specific for single-stranded nucleic acids, with a 1000-fold increase in dye fluorescence upon binding to a 20-mer ON (28Singer V.L. Jones L.J. Yue S. FASEB J. 1995; 9 (abstr.): 1422Crossref Scopus (69) Google Scholar). We also measured encapsulation of Cy3-labeled ONs in anionic liposomes by directly measuring Cy3 fluorescence and obtained identical results. Phospholipid content in the final preparations was 60–70% of the initial amount, reflecting losses during extrusion and purification by minicolumn centrifugation (Table I).Table IPhysiochemical features of anionic liposomes encapsulating ONsLipidMean diameter ± S.D.% ON encapsulated1-aMean ± S.D. of >5 independent experiments.% Phospholipid recovered1-aMean ± S.D. of >5 independent experiments.nmDOPC/DOPG216.0 ± 7556.8 ± 3.072.0 ± 6.1DOPC/DOPS242.3 ± 9046.3 ± 10.769.3 ± 7.3DOPC/DOPA229.0 ± 9244.4 ± 6.260.2 ± 8.7Lipid films with 12 mol % anionic lipid were hydrated with 75 nmol of ONs in 500 µl of 10 mm HEPES buffer (pH 7.4) with 5 mm NaCl. See “Experimental Procedures” for details on liposome preparation and analysis. Size distributions are from one representative sample.1-a Mean ± S.D. of >5 independent experiments. Open table in a new tab Lipid films with 12 mol % anionic lipid were hydrated with 75 nmol of ONs in 500 µl of 10 mm HEPES buffer (pH 7.4) with 5 mm NaCl. See “Experimental Procedures” for details on liposome preparation and analysis. Size distributions are from one representative sample. Anionic liposomes composed of DOPC with 12 mol % DOPG (DOPC/DOPG liposomes) were prepared in 10 mm HEPES buffer (pH 7.4) with 5, 50, or 150 mmNaCl, and ON encapsulation was measured. Increasing the ionic strength of the hydration buffer dramatically decreased ON encapsulation (Fig.1 B). The buffer with 5 mm NaCl was used for all subsequent studies because this allowed for maximum encapsulation. To investigate the role of anionic charge density on encapsulation, we varied the mol % of anionic lipid in liposomes. Again, increasing the anionic charge of the lipid bilayer decreased encapsulation (Fig.1 C). We also compared the encapsulation of phosphodiester ONs in anionic liposomes with that of phosphorothioate ONs, as a function of mol % DOPG. Phosphorothioate ONs were encapsulated to a lesser extent than phosphodiester Ons, and this decreased further with increasing anionic lipid content (Fig. 1 C). The ability of anionic liposomes to effectively deliver ONs to hippocampal neurons was evaluated in an in vitro model of glutamate toxicity. Neurons exposed to glutamate alone for 48 h exhibited apoptotic features such as condensed, granular soma, neurite blebbing, and fragmentation (Fig. 2A,Veh + glu). Neurons treated with 1 µm p53 AsONs delivered by anionic DOPC/DOPG liposomes retained intact processes and smooth soma after glutamate treatment, irrespective of the chemical nature of the ONs used (Fig. 2 A,AL-dAs and AL-sAs, anionic liposomes with phosphodiester and phosphorothioate p53 antisense ONs, respectively). Treatment with 0.5 and 1 µm p53 phosphodiester AsONs in DOPC/DOPG liposomes significantly increased the survival of neurons exposed to glutamate (Fig. 2 B, AL-dAs). This neuroprotection was sequence-specific as anionic liposomes with buffer alone or with 1 µm p53 scrambled ONs (Figs. 2,A and B, AL-buf andAL-dScr, respectively) were ineffective. p53 protein levels in neurons treated with glutamate and ONs in DOPC/DOPG liposomes were determined by immunoprecipitation (Figs. 2,C and D). Exposure of hippocampal neurons to 50 µm glutamate for 15 h increased p53 expression ∼4-fold relative to untreated neurons. Pretreatment of neurons with 1 µm p53 AsONs in DOPC/DOPG liposomes prevented the glutamate-induced increase in p53 protein levels by antisense-mediated down-regulation of p53 expression. In contrast, pretreatment with 1 µm scrambled oligonucleotides in anionic liposomes did not significantly alter the glutamate-induced increase in p53 expression, proving the specificity of p53 antisense sequence used in this study. The influence of liposomal lipids on the biological performance of the vector was studied by comparing the extent of neuroprotection by p53 AsONs delivered in DOPC/DOPG liposomes with that achieved by AsONs delivered (a) in liposomes where the anionic lipid DOPG was replaced with DOPS or (b) as complexes with cationic liposomes composed of DC-Chol/DOPE. DC-Chol was the model cationic lipid in our studies as it was best tolerated by neurons based on initial toxicity screens of DC-Chol, DOTAP, and commercial transfection reagents TransFastTM and Tfx-20TM (Table II). p53 antisense ONs delivered by both anionic vectors caused a dose-dependent increase in neuronal survival after glutamate exposure, whereas AsONs complexed with DC-Chol/DOPE were largely ineffective (Fig. 3A). However, greater neuroprotection was observed with p53 AsONs delivered by DOPC/DOPG liposomes compared with DOPC/DOPS liposomes at AsON doses of 0.5, 0.7, and 1 µm. To test whether the lipids themselves could exacerbate glutamate toxicity, we treated neurons with liposomes made solely of DOPG, DOPS, or DC-Chol/DOPE (without AsONs), followed by exposure to a sub-maximal dose of glutamate (10 µm). The addition of increasing amounts of DOPG did not appreciably change neuronal survival from the 71% seen after a 48-h exposure to 10 µm glutamate (Fig. 3 B). However, treatment with 40 µg of DOPS (equivalent to the amount present in liposomes for a final ON concentration of 1 µm) decreased neuronal survival to 48%. Neurons were treated with amounts of cationic lipid required to complex 1 µm ONs in +/− charge ratios (µmol of lipid/µmol of ON) of 1/2, 1.6/1, 3.2/1, and 8/1 (6.25, 20, 40, and 100 µg of DC-Chol, respectively). Only those neurons treated with 6.25 µg of DC-Chol, i.e. where the complex has a net negative charge, survived 48 h post-glutamate. Amounts of DC-Chol where the complex would be near neutral or have a net positive charge caused extensive neuronal loss.Table IIToxicity screening of commercial cationic lipidsCationic lipidLipid/ON (+/−) charge ratio% Neuronal survival2-aPercent survival compared with untreated controls; the means of two independent experiments.%DC-Chol2 /117.3DC-Chol1 /145.7DOTAP2 /1∼0DOTAP1 /128.2TransFast™2 /1∼0Tfx-20™3 /1∼0Neuronal survival was assessed 8 h after incubation with cationic lipid-ON complexes.2-a Percent survival compared with untreated controls; the means of two independent experiments. Open table in a new tab Neuronal survival was assessed 8 h after incubation with cationic lipid-ON complexes. Although p53 phosphodiester AsONs were not neuroprotective when delivered “free”, i.e. without encapsulation in anionic liposomes, free p53 phosphorothioate AsONs, at a dose of 5 µm, significantly increased neuronal survival (Fig.4A, sAs) compared with neurons treated with glutamate alone. Phosphorothioate AsONs, when delivered via DOPC/DOPG liposomes (Fig. 4 A,AL-sAs), provided significantly more neuroprotection at concentrations of 0.5 and 1 µm than 5 µmfree sAs. Neither phosphorothioate p53-scrambled ONs nor a sequence with 6 mismatches to p53 AsON were neuroprotective (Fig. 4 A,sScr and sMm, respectively, 5 µmeach). Neuronal survival was also not increased by 1 µmphosphorothioate-scrambled ON in anionic liposomes (data not shown). Phosphorothioate ONs at concentrations greater than 5 µmcaused neurons to detach from the culture substrate within 12 h of exposure and were not tested. PFT-α is a chemical inhibitor of p53 that was shown to protect cells from p53-induced apoptosis caused by genotoxic stress (29Komarov P.G. Komarova E.A. Kondratov R.V. Christov-Tselkov K. Coon J.S. Chernov M.V. Gudkov A.V. Science. 1999; 285: 1733-1737Crossref PubMed Scopus (1104) Google Scholar). Antagonists to theN-methyl-d-aspartate and α-amino-3-hydroxy- 5-methyl-4-isoxazole propionic acid glutamate receptors, MK-801 and CNQX, 20 µm each, used individually or together, and 10 µm PFT-α significantly increased the survival of glutamate-treated neurons (Fig.4 B, MK, CN, MK+CN, andPFT-α). In our hippocampal cultures, concentrations of PFT-α greater than 10 µm (20–100 µm) were toxic, whereas lower concentrations (0.5–7 µm) were not significantly protective. Neuroprotection afforded by 1 µm p53 AsON in DOPC/DOPG liposomes (Fig. 4 B,AL-dAs) was greater than that by either MK-801, CNQX, or PFT-α and comparable with that by MK-801+CNQX. After a 1-h incubation of neurons" @default.
• W2163537252 created "2016-06-24" @default.
• W2163537252 creator A5016845827 @default.
• W2163537252 creator A5042171311 @default.
• W2163537252 creator A5048322792 @default.
• W2163537252 creator A5057359171 @default.
• W2163537252 date "2001-08-01" @default.
• W2163537252 modified "2023-09-29" @default.
• W2163537252 title "Neurons Are Protected from Excitotoxic Death by p53 Antisense Oligonucleotides Delivered in Anionic Liposomes" @default.
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