oligonucleotide complexes and on their interactions with cells

Biochimica et Biophysica Acta 1390 Ž1998. 119–133

Effect of serum components on the physico-chemical properties of cationic lipidroligonucleotide complexes and on their interactions with cells Olivier Zelphati, Lisa S. Uyechi, Lee G. Barron, Francis C. Szoka Jr.

)

Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, UniÕersity of California, San Francisco, CA 94143-0446, USA Received 14 April 1997; revised 17 July 1997; accepted 29 July 1997

Abstract The interactions among serum components and cationic lipid–nucleic acid complexes are central to the understanding of how serum inhibits cellular delivery of oligonucleotides in vitro and in vivo. In this study, we show that several serum proteins, in particular bovine serum albumin ŽBSA., lipoproteins ŽHDL and LDL. and macroglobulin, interact with cationic lipidroligonucleotide complexes, alter the complex diameter and zeta potential Žfrom positive to negative values., and significantly interfere with the ability of 1,2-dioleoyl-3-trimethylammonium-propane ŽDOTAP. to deliver phosphorothioate oligonucleotides ŽODN. into cells. Serum and BSA do not dissociate the ODN and lipid components, therefore inhibition of delivery cannot be attributed to a displacement of cationic lipid from the ODN. Rather BSA at 2.5 mgrml, comparable to the amount found in 10% serum, decreases the cell association of ODN by about 5-fold and nuclear uptake of ODN by greater than 20-fold. In contrast, immunoglobulin G, the other major serum component, alters the zeta potential from positive to near neutral, has a modest effect on the diameter of the complex but does not affect cell association or nuclear delivery of the ODN at amounts found in 10% serum. Other molecules found in serum, specifically oleic acid and heparin, displace the ODN from the complex and thus interfere with delivery. This displacement is attenuated by first incubating the complex with BSA. Another manifestation of serum–complex interactions is that ODN significantly and cationic liposomes slightly, activate complement. However, formation of the complex markedly reduces the complement activation of the ODN. Finally, the effect of serum can be partially counteracted by the selection of the helper lipid ŽDOPE or cholesterol.. Inclusion of a helper lipid reduces the effective charge ratio Žcationic groupsranionic thioates. required to deliver ODN into cells and permits delivery in the presence of greater percentages of serum in the culture medium. These results support the current view that the binding of serum proteins to the complex is a significant factor in modulating the activity of cationic lipid–ODN complexes in culture and after intravenous administration. q 1998 Elsevier Science B.V. Keywords: Cationic lipid; Confocal microscopy; Fluorescence; Gene therapy; Oligonucleotide; Serum

Abbreviations: ODN, oligonucleotides; F-ODN, fluorescein-labeled oligonucleotides; DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane; DOPE, dioleoylphosphatidylethanolamine; N-Rh-PE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-ŽLissamine rhodamine B sulfonyl.; BSA, bovine serum albumin; FBS, foetal bovine serum; HDL and LDL, high and low density lipoprotein ) Corresponding author. Fax: q1 415-476-0688; E-mail: [email protected] 0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 6 9 - 0

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1. Introduction In the last decade, synthetic cationic lipids have been developed to complex with negatively charged nucleic acids and deliver them into cells w1–3x. These cationic lipid formulations deliver functionally active RNA as well as single and double strand DNA to cells. However, their delivery efficiency is reduced by the presence of serum in culture media w3–6x. Even for those cationic lipids that are claimed to be resistant to the inhibitory effects of serum on nucleic acid delivery, the serum concentrations examined are never greater than 50% w1,2x. In spite of the in vitro serum inhibitory effects, in vivo gene delivery mediated by intravenous administration of cationic lipid– DNA complexes still occurs w1x. The variability of in vivo gene delivery w1x, and the failure of cationic lipids to increase antisense oligonucleotide Ž ODN. activity in vivo w7,8x suggest the possibility of interactions between the complexes and various blood components and call into question the stability of nucleic acidrcationic lipid complexes in biological fluids. Serum components bound to the complex may lead to an increase in particle diameter and entrapment in capillary beds, poor complex accessibility to cells or increased clearance of complexes by the reticuloendothelial system ŽRES.. Although there have been extensive biological applications of lipid–ODN complexes, very few studies have been reported on the interactions between synthetic cationic lipid–ODN complexes and blood components. Senior et al. w4x reported that the incubation of cationic lipids in plasma increases turbidity and leads to the formation of clot-like masses. If large aggregates are formed, they could be physically entrapped in the lung. Indeed, Litzinger et al. w9x found that association of ODN with a cationic liposome composed of DC-chol:DOPE at a 4:1 Ž". ratio altered the pharmacokinetics and distribution of the lipid upon intravenous administration in mice. Greater than 50% of the IV administered cationic liposomes distributed to the liver and less then 10% were found in the lung within 5 min. When ODN was present, the complex lodged predominantly in the lung Ž ) 80% of the dose. and less than 10% of the dose was found in the liver at early times post-injection. At later times, the complex redistributed into the liver. The authors speculated that this was caused by a rapid

growth of the complex after injection which was mediated by interactions with serum components w9x. This putative growth in size did not occur to the same extent with the precursor cationic liposomes w9x. They suggested that a sufficiently large complex would be physically trapped in the pulmonary capillary bed w9x. Two recent studies have shown that serum complement can be activated by cationic lipids alone or when complexed with plasmid DNA w10,11x. Complement activation could result in complement components binding to the complex and targeting it to receptors for complement components that are found in the lung w12x; this could also explain the rapid clearance of cationic liposomes from the blood and uptake into the lung. Felgner and Holm w5x showed that chondroitin sulfate inhibited transfection by cationic lipid–DNA complexes and suggested that a polyanionic molecule similar to chondroitin sulfate to be the inhibitory component in serum. This suggestion received added support when it was shown that dextran sulfate and heparin can dissociate the cationic lipid from either DNA w13x or from ODN w14x. Other than the above examples, the identification of blood proteinsrcomponents that interact with cationic lipid–ODN complexes and the mechanism of serum inhibition Ždestabilization of complexes andror inhibition of cellular uptake. have not been carefully studied, whereas the factors governing the protein interactions and stability of classical liposomes have been extensively examined w15,16x. Lipid structure Žspecific lipid head group and acyl chain composition., liposome formulation and liposome charge, influence the amount and type of serum protein bound to liposomes w17,18x. Additionally, Chonn et al. w17x demonstrated a direct correlation between the amount of plasma protein bound to liposomes and their rate of elimination from systemic circulation. They identified b2-glycoprotein I to be a major protein bound to the rapidly cleared anionic liposomes w19x. On the other hand, cationic liposomes composed of stearylamine and phospholipids bind a different spectrum of serum proteins; among them are albumin, immunoglobulin and some unidentified high molecular weight proteins w18x. Studies such as these led to the realization that reducing the binding of opsonizing plasma proteins to liposomes could improve their biodistribution. This was accomplished by inclusion of cholesterol, or so-called ‘‘stealth molecules’’ such

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as gangliosides or polyethyleneglycol-modified phospholipids to increase liposome stability in blood and reduce rapid clearance w20,21x. The description of the proteins that interact with cationic lipid–ODN complexes have not yet been published but based upon the precedence in the liposome field, knowing what they are and how they interact with the complex should lead to improved formulations. In spite of many similarities in the colloidal properties between cationic lipid complexes formed with either DNA or ODN as well as similarities in the effect of serum on the interactions of the respective complex with cultured cells, there may be subtle differences in their structure that influence the interaction with serum proteins. In this study, fluorescent-labeled phosphorothioate oligonucleotides ŽF-ODN. were associated with the quaternary ammonium cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane ŽDOTAP. , and the intracellular delivery of the F-ODN was evaluated by confocal microscopy. The interactions between DOTAPrFODN complexes and serum or certain individual blood components were studied by gel retardation, fluorescence resonance energy transfer, zeta potential and size measurements. Lastly, we assessed the ability of these protein-bound complexes to activate the complement system. The results help to explain how certain serum components block ODN delivery in culture.

2. Experimental procedures 2.1. Cells and reagents CV-1 cells were cultivated in DME-H21 supplemented with 10% FBS, 2 mM non-essential amino acids, 10 mM HEPES and antibiotics Ž 100 unitsrml Penicillin, and 100 unitsrml Streptomycin. at 378C in a humidified atmosphere containing 5% CO 2 . Sheep red blood cells ŽSRBs. were purchased from Colorado Serum Ž Denver, CO. . 1,2-dioleoyl-3-trimethylammonium-propane Ž DOTAP . , dioleoylphosphatidylethanolamine ŽDOPE. and 1,2-dioleoyl-snglycero-3-phosphatidylethanolamine-N-Ž Lissamine rhodamine B sulfonyl. Ž N-Rh-PE. were obtained from Avanti Polar Lipids Ž Alabaster, AL.. Cholesterol, oleic acid, bovine serum albumin ŽBSA., high density

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lipoprotein Ž HDL. , low density lipoprotein Ž LDL. , fibrinogen, Immunoglobulin G wIgG Ž monkey rhesus.x, a-2 macroglobulin, rabbit anti-SRBs stroma and human complement sera were obtained from Sigma Ž St. Louis, MO. . Heparin was obtained from Elkins-Sinn. Ž Cherry Hill, NJ.. b2-glycoprotein was purchased from Calbiochem ŽSan Diego, CA. . 2.2. Oligonucleotides The phosphorothioate oligonucleotides used in these studies were a generous gift of G. Zon of Lynx Therapeutics Ž Foster City, CA. . A 25-mer oligonucleotide was complementary to the murine b actin messenger RNA Ž 5X-TCTGGGTCATCTTTTCACGGTTGGC-3X . and labeled at the 5X end with fluorescein ŽF-ODN.; an unlabeled antisense anti-rev 28-mer Ž 5X-TCGTCGCTGTCTCCGCTTCTTCCTGCCA-3X .. Synthesis and purification of the ODN were carried out as previously reported w22x. 2.3. Preparation of liposomes and complexes with oligonucleotides Cationic liposomes ŽDOTAP alone; DOTAP: D O PE ,1:1 Ž m olar ratio of com ponents . ; DOTAP:cholesterol, 1:1; and DOTAP:DOPE: cholesterol, 1:1:1. were prepared as previously reported w23x. Anionic liposomes composed of dioleoylphosphatidylglycerol, DOPE and dioleoylphosphatidylcholine in the molar ratio of 1:2:1 were prepared as reported w13x. For fluorescently labeled liposomes, 1 mol% of N-Rh-PE were added to DOTAP lipids. The lipidrODN complexes were prepared by adding 0.25 mg of F-ODN to different concentrations of cationic liposomes to obtain the optimum charge ratio for nuclear delivery. The charge ratio was calculated using 24 negative charges per 25-mer oligonucleotides and one positive charge per molecule of cationic DOTAP lipid. The mixture was allowed to stand at room temperature for 30 min and then the complex was transferred into DME-H21 medium with or without serum, BSA or other specified components. The size of lipidrODN complexes in the presence or absence of serum components was determined as mean "S.D. of diameter on the basis of weight average with a sub-micron particle analyzer Ž Coulter model N4 . . The zeta potential of

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DOTAPrODN complexes was determined from their electrophoretic mobility in a Zetasizer 4 Ž Malvern Instruments, UK.. 2.4. Intracellular deliÕery studies CV-1 cells were grown on coverslips and washed once in serum free medium. The cationic lipidrFODN complexes were prepared in medium as described w6x and transferred directly onto CV-1 cells. Cells were then incubated at 378C for 3 h. The oligonucleotide Ž 25-mer F-ODN. concentration was kept at 160 nM for all confocal imaging experiments. The lipid concentration varied due to the required charge ratio, resulting in lipid concentrations between 14 and 38 mM. After incubation, the CV-1 cells were washed three times in PBS and coverslips were immediately mounted on hanging drop slides Ž Fisher Scientific, Pittsburgh, PA.. Finally, cells were directly observed by confocal microscopy. 2.5. Confocal laser scanning microscopy A MRC-600 ŽBiorad, Richmond, CA. confocal laser scanning imaging system equipped with a kryptonrargon mixed gas laser, upright microscope ŽNikon, NY. and a 60 = oil immersion objective lens ŽNikon, NY. was used. A single filter block for fluorescein at 488 nm was used to monitor fluorescence emission. To prevent photobleaching, the confocal microscope was operated under conservative laser intensity and time exposure conditions. The COMOS confocal software program ŽBiorad, Richmond, CA. was employed to control the confocal module’s functions and to analyze the images. 2.6. Fluorescence resonance energy transfer (FRET) Fluorescence was measured with a SPEX Fluorolog 2 spectrophotometer ŽSpex Industries, Edison, NJ. as previously described w24x. For the characterization of FRET, unlabeled or F-ODN were used at 0.5 mgrml in 2 ml of Tris–HCl buffer Ž30 mM Tris– HCl, pH: 7.5. or NaCl buffer Ž150 mM NaCl, 2 mM MgCl 2 , 2 mM CaCl 2 and 10 mM Tris, pH: 7.4. and DOTAP:N-Rh-PE liposomes were mixed with ODN

at a 10 to 1 Ž". charge ratio relative to the positive charge on DOTAP per negative charge on the ODN. Emission spectra were recorded between 500 and 600 nm with excitation at 470 nm. Fluorescein fluorescence quenching Ž Q . was calculated as Q s Ž 1 y w FŽdonorq acceptor.rFŽdonor alone. x. )100, where F s fluorescence level at 520 nm. In the presence of serum components, fluorescein fluorescence dequenching Ž d Q . was calculated as d Q s Q 1Ž complexes . y Q 2 Ž complexes q compounds . , where Q1 represents maximum quenching. Different concentrations of serum components were added to the complexes and emission spectra were recorded as above. 2.7. Gel electrophoresis experiments The preparation of the DOTAPrODN complexes was carried out in 96 well plates as follows: 1 mg of ODN was added to DOTAP at a charge ratio Ž". of 10 to 1. The mixture was allowed to stand at room temperature for 30 min, and the serum component to be examined was added to the complexes. After 15 min, 10 ml of 30% glycerol were added and the mixture was loaded on a 20% non-denaturing polyacrylamide gel. A potential of 150 V was applied for 4 h. Oligonucleotides were detected by staining the gel with SYBR Green I. 2.8. Complement assay The principle of the assay is based on the formation of the membrane attack complex which is induced when antibody-sensitized sheep red blood cells ŽSRBs. are incubated with serum. The membrane attack complex lyses the erythrocytes and the released hemoglobin is measured by absorption with a spectrophotometer. This assay was performed as described w11x. The volume of serum which causes the lysis of 50% of the erythrocytes is designated as the CH50. For each compound, the effect of various serum concentrations on the CH50 value was compared to the CH50max, which is the maximum volume required when SRBs are incubated with untreated serum. The results were presented as % CH50maxs ŽCH50 samplerCH50max. = 100.

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3. Results 3.1. Effect of serum components on ODN nuclear uptake mediated by cationic DOTAP lipids Cationic liposomes have been shown to modify the intracellular localization of ODN. When delivered alone, the ODN are localized in punctate cytoplasmic regions of the cell, but when delivered as a cationic lipid complex the ODN are also observed within the nucleus w6,25,26x. This localization of F-ODN was found to depend on the charge ratio Ž". used to form the complex. Since microinjected F-ODN rapidly accumulates into nuclei w24,27x, we interpret this localization as release of ODN from the complex into the cytoplasm. The optimum charge ratio for delivery by DOTAP liposomes was determined to be 10 to 1 w6x. We examined the effects of serum and of BSA, the most abundant serum protein, on the F-ODN delivery mediated by cationic DOTAP lipids. In the absence of serum or BSA, 50–70% of cells exhibited a fluorescent nucleus ŽFig. 1ŽA.., confirming that FODN delivered by DOTAP were localized as previously described w6x. When increasing amounts of serum or BSA were added to the medium, the nuclear localization of ODN was strongly depressed. The presence of either 2.5% serum or of 0.6 mgrml of BSA Žwhich corresponds to the BSA concentration in 2.5% serum. completely blocked nuclear delivery ŽFig. 1ŽA... Quantification of total fluorescence intensity Žtotal pixel intensityrfield. approximates the total cell-associated F-ODN and showed a large reduction of fluorescence intensity in the presence of at least 2.5% of serum ŽFig. 1ŽB... Thus the presence of serum Ž Fig. 1Ž B.. or of BSA Ž Fig. 1Ž A. , Fig. 4. , a major serum component, inhibited the nuclear delivery of F-ODN mediated by DOTAP, and coincided with a depression in total F-ODN association with the cell. The potential for other serum components to block the ODN uptake mediated by DOTAP was also tested. In previous studies lipoprotein, globulin and immunoglobulin have been shown to bind liposomes and alter liposomal structure and integrity w15,16x. At low concentrations, LDL, HDL, a 2-macroglobulin, fibrinogen and heparin completely inhibit nuclear uptake of F-ODN when it was delivered by the complex ŽTable 1.. This demonstrated that several blood compounds

Fig. 1. Effects of serum, BSA and IgG on F-ODN nuclear uptake mediated by DOTAP. DOTAP liposomes were complexed with F-ODN at a 10r1 Ž". charge ratio. CV-1 cells were incubated for 3 h with the complexes at 378C in media containing FBS, BSA or IgG. Cells were examined using the confocal microscope as described in Section 2. ŽA. The percentage of total cells exhibiting nuclear fluorescence in the presence of serum or BSA. Ž ) . Corresponding concentrations are based on 24 mgrml of BSA as equivalent to BSA in 100% serum. Each bar represents the mean of three separate experiments, and the S.D. did not exceed 10%. ŽB. The total fluorescence intensity per field was determined with the COMOS confocal software program and represents the mean of five different fields Ž15–25 cellsrfield.. ŽC. The percentage of cells exhibiting nuclear fluorescence in the presence of IgG. Each bar represents the average over three fields of cells Ž G 20 cellsrfield..

can interact with positively charged DOTAPrODN complexes and block DOTAP mediated ODN delivery. In contrast, IgG and b2-glycoprotein showed no significant effect on nuclear uptake of ODN at levels

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Table 1 Effect of various blood components on the delivery efficiency and the stability of DOTAPrODN complexes

Serum free media 10% FBS BSA Heparin Oleic acid a 2-Macroglobulin Fibrinogen HDL LDL IgG b2-Glycoprotein I

Normal concentration in human plasma

Isoelectric point

Concentration in medium

% of labeled nuclei a

% of fluorescein dequenching a

N.A.b N.A.b 35–45 mgrml N.A. N.A. 2.2–3.8 mgrml 2.9 mgrml 3.7–11.7 mgrml 4.1–6.7 mgrml 12–18 mgrml 0.2–0.25 mgrml

N.A.b N.A.b 4.9 - 2.0 - 5.5 5.4

N.A.b 7.5 mgrml 2.4 mgrml 15 unitsrml 1.5 mmolesrml 100 mgrml 300 mgrml 350 mgrml 350 mgrml 1 mgrml 20 mgrml

65.2 " 2.8 0 0 0 Cytotoxic c 0 0 0 0 59.2 " 2.2 56.55 " 4.3

N.A.b Not determined 4 " 0.8 69.9 " 8.8 52.8 " 0.8 0.6 " 0.1 2 " 0.1 6.6 " 0.1 3.2 " 1.5 4.1 " 2.6 0.2 " 0.3

d d d

5.8–7.3 d

a

Mean and range of two experiments. Where there are zeroes in the table, no fluorescent nuclei were detected. Not applicable. c At non-toxic concentration Ž0.37 mmolrml., 29.8% of nuclear uptake was observed. d Not precisely known. b

found in 10% serum Ž Table 1. . Immunoglobulin G did not decrease nuclear uptake until concentrations that were similar to those found in complete serum are reached Ž 10 mgrml. ŽFig. 1ŽC... At this concentration, nuclear uptake was reduced about 4-fold and cell-associated fluorescence was reduced about 2-fold ŽFig. 1ŽC... Oleic acid, a non-esterified fatty acid, also inhibited the ODN nuclear uptake by 50% at a concentration of 0.37 mmolrml. Significant toxicity was observed at concentrations greater than 0.75 mmolrml, therefore the decrease in nuclear localization is confounded by the toxic effects of the compound. 3.2. Effect of serum components on the stability of DOTAPr ODN complexes One potential explanation for the serum inhibition is that the complex dissociates when the serum component interacts with it. If this occurs, the cationic lipid would be unable to deliver the ODN. We have studied the stability of the complexes in the presence of serum and certain serum components by fluorescence resonance energy transfer Ž FRET. and gel electrophoresis. The formation and dissociation of complexes was monitored by the FRET between donor F-ODN and acceptor DOTAP:N-Rh-PE liposomes w13x. When in close proximity, the fluorescein emis-

sion ŽF-ODN. was specifically quenched ŽFig. 2Ž A.. and rhodamine emission ŽDOTAP:N-Rh-PE. was increased. When the donor and acceptor were separated the fluorescein becomes ‘‘dequenched’’ and the energy transfer is reversed. Heparin and oleic acid reversed the FRET Ž Fig. Ž 2 A. and Table 1. almost to the levels observed with F-ODN alone. This displacement of F-ODN from the complex was dependent on the concentrations of heparin and oleic acid Ž data not shown. . The release of F-ODN from the complexes was rapid and the fluorescein dequenching reached a plateau between 3 and 5 min Ždata not shown. . In contrast, BSA, a 2macroglobulin, fibrinogen, HDL, LDL, IgG and b2glycoprotein, were unable to reverse the energy transfer ŽFig. 2Ž A. and Table 1. . This indicated that the ODN remained associated with the liposome complex ŽDOTAP:N-Rh-PE.. Due to the high background fluorescence of serum, we were unable to apply the FRET assay to determine if the presence of serum could separate ODN from cationic lipids. Release of oligonucleotides from the complexes was confirmed by a gel shift assay ŽFig. 2ŽB... With DOTAP complexes prepared at a 10 to 1 charge ratio Ž". , ODN migration was completely retarded compared to ODN alone. When proteins, oleic acid or heparin were included in the mixture applied to the gel, band distortion occurred, none-the-less when

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Fig. 2. Effects of BSA, heparin and oleic acid on DOTAPrODN complex stability. ŽA. FRET: BSA, heparin and oleic acids were added to preformed complexes prepared at 10r1 Ž". charge ratio and the efficiency of energy transfer was recorded by scanning the emission spectra from 500 to 600 nm with excitation at 470 nm. ŽB. Gel retardation: Individual compounds were mixed with complexes prepared at 10r1 Ž". charge ratio for 15 min and run on a 20% non-denaturing polyacrylamide gel. Lane 1 and 10: ODN alone, lane 2 and 7: DOTAPrODN, lane 3 and 8: oleic acid Ž0.6 mmol., lane 4: BSA Ž1 mg., lane 5: heparin Ž15 units., lane 6: 10% serum containing medium, and lane 9: BSA Ž1 mg. q oleic acid Ž0.6 mmol..

ODN was released from the complex, it clearly migrated into the gel Ž Fig. 2Ž B.. . Addition of heparin or oleic acid caused ODN release, agreeing with the FRET dequenching data. In contrast, ODN was retained at the origin in the presence of serum or BSA. The pattern of ODN migration on the gel directly confirmed the FRET studies in which heparin and oleic acid were able to displace F-ODN from cationic lipid complexes, whereas other compounds and serum do not exhibit this displacement effect.

3.3. Effect of BSA on the integrity of DOTAPr ODN complexes Since BSA is known to bind and act as a carrier of fatty acids w28x, we asked whether the presence of BSA would affect complex destabilization by heparin or oleic acid. As increasing concentrations of BSA were preincubated with the complexes, the displacement of ODN by oleic acid and heparin was markedly reduced ŽFig. 3.. Oleic acid was more sensitive to the

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serum, HDL Žat 350 mgrml. protected the complex from destabilization, but b 2-glycoprotein Ž at 20 mgrml. did not markedly modify the destabilization due to heparin Ždata not shown. . These data suggested that despite the dissociationrdestabilization that occurs with individual serum components, other serum components such as HDL and BSA can prevent the release of ODN from the cationic complexes. 3.4. Effect of serum and BSA on interaction of DOTAPr F-ODN complexes with cell membranes Fig. 3. Effect of BSA on the heparin and oleic acid mediated destabilization of DOTAPrF-ODN complexes. Increasing concentrations of BSA were added to preformed complexes prepared at 10r1 Ž". charge ratio for 15 min. Then, oleic acid or heparin were added to the mixtures and the efficiency of energy transfer was recorded by scanning the emission spectra from 500 to 600 nm with excitation at 470 nm. Values are the mean of duplicate measurements that agreed to within "5%.

protective effect of BSA, since much less BSA was required Ž 1 to 20 molar ratio of BSArfatty acid. than with heparin Ž10 to 1, BSArheparin. to protect the complex from dissociation. These results were confirmed by gel retardation assays. For complexes incubated with BSA prior to addition of heparin or oleic acid, no band that co-migrated with free ODN was observed Ž Fig. 2ŽB., lane 9.. Similarly, prior incubation of complex with serum prevented the destabilization associated with heparin and oleic acid Ždata not shown.. Thus the interaction of cationic lipidrODN complexes with BSA was able to protect the complex from the dissociating action of heparin and oleic acid. b2-glycoprotein and lipoproteins have been reported to bind heparin w29,30x. Therefore we tested the ability of the b2-glycoprotein and HDL to protect the complex from the heparin-induced destabilization. At the concentrations corresponding to values in 10%

Another possible inhibitory mechanism to delivery of the ODN by the cationic lipids is the binding of serum components to the complex, preventing their interactions at the cell surface or hindering cellular internalization. To investigate these possibilities, DOTAPrF-ODN complexes were added to CV-1 cells in the presence or absence of 10% of serum or BSA. After 1 h at 48C, the cells were rinsed with serum free or BSA free medium to remove complexes that were not bound to the cell and the cells were incubated for another 2 h at 378C. For complexes preincubated with 10% serum, the nuclear localization of F-ODN was completely inhibited Ž Fig. 4ŽA. and ŽC... When BSA Ž 1.2 or 2.4 mgrml. was included in the preincubation medium, 29% and 10% of cells exhibited nuclear fluorescence which was a decrease from the 62% fluorescent nuclei observed in serum free medium Ž Fig. 4Ž A. and Ž B... Analysis of the fluorescence images of cells incubated in the presence of protein also showed a lower fluorescence intensity per field ŽFig. 4Ž A.. , indicating a reduction of complex on the cell surface andror internalized within the cell. To determine if prior occupancy of surfacercell membrane ‘‘binding sites’’ could inhibit subsequent

Fig. 4. Effects of serum and BSA on the association DOTAPrF-ODN complexes with cells. ŽA. F-ODN Ž140 nM. complexed to cationic DOTAP lipids Ž38 mM. at a 10r1 charge ratio Ž". were incubated, in serum free medium with and without BSA or in 10% serum containing medium, on CV-1 cells growing on cover slips. After 1 h at 48C, the medium was removed and cells were incubated 2 h in serum free medium at 378C. Coverslips were mounted, after rinsing in PBS, on hanging drop slides and observed by confocal microscopy as per the normal procedure. The percentage of nuclear fluorescence represents the mean of three separate experiments and the total fluorescence intensityrfield represents the mean of five different fields. ŽB. Nuclear uptake of F-ODN delivered as a 10:1 Ž". complex in serum free medium, as visualized by confocal microscopy. The left panel corresponds to the direct transmitted light image of CV-1 cells. ŽC. Complexes were incubated as described above in medium containing 10% of FBS. The left panel corresponds to the direct transmitted light image, and the right panel is the confocal microscopy image.

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delivery of F-ODN, unlabeled complexes were incubated with CV-1 cells before the addition of labeled complex. After 1 h of incubation Žat 48C. in the presence or absence of 10% serum supplemented media, the unassociated complexes were removed and DOTAPrF-ODN labeled complexes were added in serum free medium for 2 h at 378C. Preincubation with unlabeled complex in the absence of serum resulted in a marked decrease in the subsequent delivery of fluorescent complex Ž data not shown. . 3.5. Effect of serum components on the size and zeta potential of the complex Although nuclear fluorescence was blocked in the presence of serum or BSA, fluorescent complexes were still cell associated Ž Fig. 4Ž C.. , but the fluorescence intensity per field was significantly diminished ŽFig. 4ŽA.. . This suggested that serum components interfered with the surface interactions and also with the uptake pathway Žendocytosis. of the complex. Interactions of serum components with the complex could alter their charge and size. This could significantly affect the cell association and uptake into and escape from the endosome. We measured the zeta potential Ž net surface charge density. and the diameter of the DOTAPrODN complexes before and after their association with various serum components. In the absence of any components, the diameter of the complex was 0.110 " 0.030 mm and the zeta potential was 13 " 3 mV. Heparin at 15 unitsrml decreased the zeta potential of the complex to y41 " 2 mV but did not alter the diameter. HDL at 350 mgrml slightly decreased the zeta potential to 7 " 3 mV and caused a dramatic aggregation of the complex. The particle diameters ranged from 1.5 to 8.5 mm and were very polydisperse. BSA and IgG had considerably different effects on the complex ŽFig. 5.. As the concentration of BSA in the incubation medium increased, there was a reversal of the zeta potential from positive to negative ŽFig. 5ŽA.. . There was also an increase in particle diameter from about 0.14 mm in the absence of BSA to about 1.5 mm at 24 mgrml Ž Fig. 5Ž A.. . The polydispersity also increased up to 24 mgrml BSA concentration. At 35 mgrml BSA, the zeta potential remained negative but the particle diameter was not significantly different than that of the starting com-

Fig. 5. Effects of BSA and IgG on DOTAPrODN complex mean diameter and zeta potential. The mean particle diameter Žsolid bar. and zeta potential Žline with grey circles. of liposomes alone Žno ODN., complex alone Žno protein., or of complex in the presence of various concentrations of ŽA. BSA or ŽB. IgG. The complexes prepared at a 10r1 Ž". charge ratio. ODN concentration was kept constant at 0.75 mM Žfinal concentration during incubation with protein.. The zeta potential was the mean of six measurements and had an S.D.F"3 mV. The particle diameter which was the mean of three measurements had an S.D. "80% at BSA concentrations of 12 and 24 mgrml and an S.D.F 30% at all other protein concentrations for both BSA and IgG, and in the absence of protein.

plex ŽFig. 5ŽA... IgG up to a concentration of 3 mgrml reduced the zeta potential to 2 " 2 mV and caused a slight decrease in the particle diameter. At 10 mgrml IgG, the zeta potential remained positive but the diameter increased to 0.6 mm and became more polydisperse ŽFig. 5Ž B.. . Compounds that inhibited ODN nuclear delivery either imparted a negative charge on the complexes ŽBSA and heparin. or considerably increased their size ŽBSA, HDL. . In contrast, at conditions that do not inhibit the ODN nuclear uptake ŽBSA at 0.24 mgrml and IgG at 3.0 mgrml. the complexes still exhibit a positive charge density and their size is

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not markedly changed ŽTable 1, Fig. 5.. These results demonstrated that the serum mediated inhibition of uptake was coincident with changes in the complex size and charge. This suggested that interactions with cell membranes are not solely based on electrostatic interactions and that internalization becomes limited with increased complex size. The inhibitory effect of BSA on DOTAP-mediated ODN nuclear uptake may also be attributed to interference with the mechanism of ODN dissociation from cationic lipids and their subsequent cytoplasmic release. Indeed, we have recently demonstrated that anionic phospholipids present in vesicular membranes caused a rapid release of oligonucleotides from the complex after their internalization in cells w14x. We have studied the effect of BSA on the ability of anionic liposomes Ž dioleoylphosphatidylglycerol: DOPE:dioleoylphosphatidylcholine, molar ratio 1:2:1. to displace the F-ODN from cationic DOTAP lipids by FRET. The preincubation of the complex with BSA markedly reduced the effect of anionic liposomes in release of the F-ODN from DOTAP Ždata not shown.. This suggests that serum proteins, particularly BSA, can also interfere with the intracellular release of F-ODN. 3.6. ActiÕation of the complement system by ODN, DOTAP and DOTAPr ODN complexes Components of the complement system may be involved in the rapid blood clearance of nucleic acidrcationic lipid complexes. Recently, Plank et al. w11x have shown that complement activation is a potentially limiting factor for gene delivery by synthetic cationic molecules since these agents were able to activate the complement system to varying extents. However, such studies have not been reported with single strand DNA or with complexes of phosphorothioate oligonucleotides and cationic lipids. We examined the effects of backbone-modified phosphorothioate ODN alone; a strong and concentration-dependent activation of complement was observed ŽFig. 6. . The CH50 value for ODN was approximately 0.1 mM. The in vitro activation of complement by ODN was not surprising since it had been reported that intravenous infusion of various ODN Ždifferent sequences. resulted in complement activation w31x. DOTAP alone and when complexed

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Fig. 6. Complement activation of ODN, DOTAP lipids and DOTAPrODN complexes. Various concentrations of ODN alone or complexed with DOTAP lipids at 10r1 Ž". charge ratio were tested as described w11x. Ž ) . DOTAP lipids alone were tested at the same concentrations used for ODNrDOTAP complexes. The concentration of DOTAP positive charge used varies from 0.2 to 500 mM. Filled circles, ODN; open squares, DOTAP; filled triangles, DOTAPrODN complex.

with ODN at a 10 to 1 Ž". charge ratio showed less complement activation than did the ODN alone Ž Fig. 6.. Comparison of the CH50 curve midpoints indicated a 35-fold decrease in complement activation for DOTAP alone and DOTAPrODN Ž midpoints between 62 and 125 mM of positive charges, or 3.7 mM of ODN.. Complement activation is reduced 2-fold when DOTAP is complexed with ODN. Thus, when ODN is complexed with cationic lipid complement activation is reduced from levels observed with individual components of the complex. 3.7. Effects of serum and BSA on ODN nuclear uptake mediated by DOTAP and ‘‘helper’’ lipids The association of neutral or ‘‘helper’’ lipids ŽDOPE and cholesterol. with DOTAP reduces the optimum charge ratio required for efficient ODN delivery w6x. Indeed, the association of DOPE and cholesterol with DOTAP yielded approximately the same degree of nuclear uptake at different charge ratios Ž 10r1 for DOTAP, 5r1 for DOTAP:cholesterol, 2.5r1 for DOTAP:DOPE and 1.25r1 for

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Fig. 7. Effects of serum on F-ODN nuclear uptake mediated by DOTAP with DOPE andror cholesterol. Various liposome formulations were complexed with F-ODN at their optimum effective charge ratio; F-O D N r D O T A P at 10r 1, FODNrDOTAP:cholesterol at 5r1, F-ODNrDOTAP:DOPE at 2.5r1 and F-ODNrDOTAP:DOPE:cholesterol at 1.25r1. CV-1 cells were incubated for 3 h with complexes at 378C in media containing varying percentages of FBS and were examined using the confocal microscope as described in Section 2. ŽA. The percentage of nuclear fluorescence represents the mean of three separate experiments and the S.D. did not exceed 15%. ŽB. The total fluorescence intensity rfield represents the mean of three different fields and the S.D. did not exceed 15%.

DOTAP:DOPE:cholesterol. . The delivery of ODN into cells by DOTAP, DOTAP:DOPE and DOTAP: cholesterol was strongly inhibited in the presence of serum ŽFig. 7Ž A.. . The inhibition by serum was concentration and formulation dependent. In 10% serum the most efficient liposome formulation was DOTAP:DOPE:cholesterol at 1.25r1 Ž". charge ratio since 35–40% of cells exhibited a fluorescentoligonucleotide labeled nucleus whereas nuclear fluo-

rescence was greatly reduced or abolished with the other formulations ŽFig. 7ŽA.. . The order of efficiency in the presence of serum for the different formulations was DOTAP:DOPE s DOTAP: cholesterol) DOTAP ŽFig. 7ŽA... Similar results were obtained when BSA was added to the culture medium Ždata not shown.. Thus including neutral lipids in the formulation permitted the use of a lower charge ratio and decreased the inhibitory effect of serum. One consequence of the inclusion of a helper lipid with DOTAP Ž which reduces the positive charge density of the complex. was that the fluorescence intensity per visual field was not significantly reduced by the presence of serum in comparison to DOTAP alone ŽFig. 7ŽB... Even though the complexes were able to bind to the cell surface in presence of 10% of serum, the F-ODN nuclear delivery mediated by DOTAP:cholesterol and DOTAP:DOPE liposomes was significantly reduced ŽFig. 7Ž A. and ŽB... This strongly suggests that other steps in the delivery process such as internalization or cytoplasmic ODN release are blocked by the presence of serum proteins on the complexes. We have also tested these different liposome formulations alone or complexed with ODN in the complement activation assay. M ixtures of D O T A P :c h o le ste ro l, D O T A P :D O P E a n d DOTAP:DOPE:cholesterol lysed the sensitized SRBs at the higher concentrations Ždata not shown. . Therefore, a quantitative measurement of complex-mediated complement activation cannot be made with this assay because we cannot measure the upper end of the lysis curve. Nevertheless, at low positive charge concentrations ŽF 62mM. no complement activation was observed with DOTAP:cholesterol and DOTAP:DOPE. The complexation of phosphorothioate ODN with these lipid compositions also strongly reduced the complement activation Ž data not shown.. However, with DOTAP:DOPE:cholesterol the activation of complement was similar to the ODN alone Ždata not shown..

4. Discussion Cationic liposomes are a valuable ODN and nucleic acid delivery reagent in vitro but have had more

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modest success for enhancing delivery after in vivo administration. This is partially due to the dramatic and yet uncharacterized effect of serum on nucleic acid delivery efficiency w3–6x. We have shown that serum and various components found in serum including BSA, lipoproteins, fibrinogen, heparin, fatty acids and a 2-macroglobulin prevent ODN delivery mediated by cationic DOTAP lipid formulations. Two obvious explanations for serum inhibition come to mind: coating of the complex with serum components and dissociation of the complex by serum components. The first explanation for the inhibition of delivery relates to non-specific interactions between the cationic lipids and serum proteins. This interaction leads to neutralization of the positive charges andror an increase in size of the complex. The results presented here suggest a polyelectrolyte charge interaction occurs between the positively charged surface of the complex and negatively charged serum components. We have studied several aspects of this serum effect by using purified components that represent both proteins that are in the highest concentration as well as proteins that previously have been identified to interact with liposomes. The results indicate that non-specific interactions with serum components alter the size and surface charge and cause the complex to aggregate. Subsequent binding of the complex to the cell surface, internalization and possibly intracellular release of ODN from cationic lipids are inhibited. In the case of BSA, the inhibition of ODN delivery mediated by DOTAP occurs at multiple steps of the cellular uptake pathway. First, the incubation of the DOTAPrODN complex with BSA leads to a particle with a more negative surface potential and a larger diameter. This results in a decrease in electrostatic interactions with cell membranes and less complex is associated with the cells. Although negatively charged particulates can bind to cell surfaces as evidenced by internalization of anionic liposomes w32,33x, the number of binding sites is limited. Although BSA associated complexes are bound to cell surfaces, the apparent diameter of the punctate fluorescence areas that are cell associated become much larger in the presence of BSA or serum. Their relatively large size very likely becomes a barrier to their internalization. Also, the presence of BSA on the surface of the complex may act as a steric barrier to prevent endo-

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somal destabilization andror ODN release from the endosome by interfering with the ability of anionic lipids to form an ion pair with the cationic lipid; a mechanism proposed to be involved in DNA w13x and ODN w14x release from the complex. This possibility is supported by the finding that pre-incubating the complex with BSA interferes with heparin-induced dissociation of the complex Ž Fig. 3. . Thus the data suggest that serum components act on several steps of the internalization pathway followed by the complex into the cell, including interactions with the cell membrane Ž due to charge neutralization., internalization Ždue to the size. and ODN intracellular release Ždue to steric hindrance.. Albumin, due to its high concentration in serum and low isoelectric point, is an obvious candidate for a major role in inhibition of ODN delivery by cationic lipid complexes. The family of IgG proteins is the second most prevalent protein species in serum. Immunoglobulins bind to negatively charged ODN w34x and also to certain liposomes w35,36x but had no effect on the delivery of the positively charged complexes at concentrations that are equivalent to 30% serum. At concentrations found in serum, IgG reduced nuclear uptake by 4-fold. Thus when compared to BSA, lipoproteins, a 2-macroglobulin or fibrinogen Ž Table 1., IgG has only a modest effect on the interaction of the complex with cells in culture. With regard to b2-glycoprotein, a major protein implicated with the binding to and rapid clearance of anionic liposomes w19x, no inhibition of nuclear uptake was observed at b2-glycoprotein concentrations found in serum. Thus, with a few notable exceptions, a variety of serum proteins can block nuclear uptake of cationic lipid delivered ODN at concentrations found in cell culture. The complement system is a collection of serum proteins that play a significant role in liposome disposition w37–39x. The finding of complement activation indicates that interactions of cationic complexes with the complement system are an additional potential limitation for ODN delivery mediated by cationic lipids. We have previously shown that complexes of plasmid DNA and DOTAP are less potent activators of complement than are cationic polymers Ž polylysine, dendrimer, polyethyleneimine. or multivalent cationic lipids ŽDOGS. w11x. In the former study, plasmid DNA, a weak activator of complement, con-

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siderably reduced complement activation by the carrier when the complex is formed. Here we show the composition of the carrier determines whether the complex can have a protective effect for the ODN. DOTAP strongly reduced the complement activation of the phosphorothioate ODN, however, the best lipid composition for ODN delivery in the presence of serum ŽDOTAP:DOPE:cholesterol. did not reduce complement activation when combined with the ODN. The reason for this observation is not known but could be related to the ability of cholesterol to enhance complement activation w10x. The effect of lipid composition in the protection against complement activation by the ODN serves as a reminder of how complicated formulation issues can become when dealing with the cationic lipids; liposome formulations designed to overcome interactions with blood components to improve ODN delivery may not be adequate for in vivo applications due to adverse interactions with other systems. In addition to interference with delivery via surface associate proteins, certain serum components, heparin and oleic acid, can dissociate the DOTAPrODN complexes. This raises the possibility that proteoglycans and fatty acids found in serum or the extracellular regions of the body might interfere with ODN delivery in vivo by bringing about release of nucleic acids before the complex can reach the target cell. Evidence has been presented for both the potential inhibitory effects of proteoglycans w5,13,14x as well as the possibility that proteoglycans may be involved in enhancing cation mediated gene transfer by providing a binding site on the cell surface for the cationic complexes w40x. However, the fact that BSA, the major serum protein, could reduce the heparin and oleic acid dissociation of the complex complicates any interpretation on the effects of these molecules in the serum inhibition mechanism. The finding that BSA can protect the complex against dissociation from certain polyanions also suggests the possibility of coating the pre-formed cationic complex with an anionic substance like BSA to protect it from interacting with other species. As shown in Fig. 5, when the concentration of BSA was 35 mgrml the complex had a net negative surface charge but maintained a diameter under 200 nm. Optimization of the coating process so that targeting ligands are included in the process might permit the

formation of a targeted negatively charged complex that can attach to cells via a ligand-mediated interaction with a cell surface receptor. In summary, we show that serum components can decrease ODN delivery into cells by changing the size and surface properties of the complex and by dissociating the complex. Serum albumin decrease cell interactions via the first modes whereas heparin can interfere with ODN delivery by dissociating the complex. In complete serum the complex remains intact, so it appears that changes in the size and charge of the complex are the principal reasons for the decreased ODN delivery efficiency in the presence of serum.

Acknowledgements This work was supported by GLAXO-Wellcome ŽOZ. and The State of California Biotechnology Research and Education Program Ž LGB. . Additional funding for this project was provided by the NIGMS Biotechnology Training Grant Ž LSU. and NIH DK46052 ŽFCS. , and the Gene Therapy Core Center ŽDK47766.. FCS has a financial interest in and serves as a consultant to GeneMedicine, a biotechnology company developing gene medicines. We acknowledge Christian Plank for the complement assay technical assistance. We thank Wayne Hendren for helpful comments and encouragement during these studies.

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