Cytosolic Delivery of Macromolecules. 3. Synthesis and

Bioconjugate Chem. 2004, 15, 1166−1173

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ARTICLES Cytosolic Delivery of Macromolecules. 3. Synthesis and Characterization of Acid-Sensitive Bis-Detergents Aravind Asokan and Moo J. Cho* Division of Drug Delivery & Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. Received May 20, 2004; Revised Manuscript Received August 9, 2004

A serious limitation that precludes utilization of single-tailed, pH-sensitive detergents for the cytosolic delivery of macromolecules is their low limit of incorporation in stable liposomal formulations. To address this issue, we have prepared two Gemini surfactants or ‘bis-detergents’ by cross-linking the headgroups of single-tailed, tertiary amine detergents through oxyethylene (BD1) or acid-labile acetal (BD2) moieties. The membrane-bound pKa of the twin tertiary amine headgroups was determined to be 6.37 ( 0.36 using a fluorescence-based assay. As evidenced by thin-layer chromatography, BD2 was hydrolyzed under acidic conditions (pH 5.0) with an approximate half-life of 3 h at 37 °C, while BD1 remained stable. Low pH-induced collapse of liposomes containing acid-labile BD2 into micelles was more facile than that of BD1. With BD1, the process appeared to be reversible in that aggregation of micelles was observed at basic pH. The irreversible lamellar-to-micellar transition observed with BD2-containing liposomes can possibly be attributed to acid-catalyzed hydrolysis of the acetal crosslinker, which generates two detergent monomers within the bilayer. Liposomes composed of 75 mol % bis-detergent and 25 mol % phosphatidylcholine were readily prepared and could entrap macromolecules such as polyanionic dextran of MW 40 kDa with moderate efficiency. The ability of BD2-containing liposomes to promote efficient cytosolic delivery of antisense oligonucleotides was confirmed by (a) their diffuse intracellular distribution seen in fluorescence micrographs, and (b) the up-regulation of luciferase in an antisense functional assay. The low pH-responsive, bis-detergent constructs described herein are suitable for triggered release strategies targeted to acidic intracellular or interstitial environments.

INTRODUCTION

Lipid-based drug carriers often promote membrane fusion or permeabilization by virtue of their inherent bilayer-perturbing conformations. As a result, incorporation of sufficient concentrations of such lipids in stable liposomal formulations poses a formidable challenge. For example, the utility of fusogenic lipids such as dioleoylphosphatidylethanolamine (DOPE) that preferentially adopt the hexagonal phase has been compromised by their low limit of incorporation into liposomal bilayers (1-3). To address this issue, Guo and Szoka (4) have devised an acid-cleavable ortho ester-poly(ethylene glycol) (PEG) conjugate of distearoyl glycerol. The PEGylated lipid not only serves to stabilize DOPE in bilayer vesicles, but also prolongs circulation half-life of the liposomal formulation in vivo. Cleavage of PEG in an acidic environment promotes lamellar-to-hexagonal transition of the liposomal formulation, which in turn results in release of contents and membrane fusion. A similar problem pertaining to liposomal incorporation of micelle-favoring, pH-sensitive detergents remains unsolved (5). Liposomes containing detergent-to-phospholipid ratios greater than 1:3 are inherently unstable * Corresponding author: Moo J. Cho, CB # 7360, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, tel. no: 919-966-1345, fax no: 919-9667778, e-mail: [email protected].

(6, 7). Stabilization of such formulations with bilayerfavoring lipids such as cholesterol is counterproductive, in that it compromises their delivery potential (5, 8). As a result, concentrations of pH-sensitive detergents required to promote efficient liposomal collapse and permeabilization of the endosomal membrane remain unattainably high (9). A chemical solution to this problem is to mask the membrane-disrupting ability of such detergents by conjugating a second lipid tail to the detergent headgroup (10) or cross-linking two single-tailed detergents with a cleavable moiety (11). Unlike conventional detergents, the latter bis-detergents (BD) or Gemini surfactants tend to behave more like phospholipids than surfactants (12). Hence, while single-chain detergents tend to adopt micellar structures, bis-detergents due to their cylindrical shape can be incorporated with relative ease in vesicle bilayers (11, 13). More importantly, such constructs allow for in situ generation of single-tailed detergents within stable bilayers upon exposure to specific stimuli, acidic pH in the present case. On the basis of the above rationale, we have developed a two-step strategy for the cytosolic delivery of macromolecules using cleavable detergent dimers that possess an acid-labile acetal linker between their polar headgroups (Figure 1). Ortho ester, acetal, and vinyl ether groups are stable under neutral conditions but hydrolyze rapidly at acidic pH due to stabilization of the intermedi-

10.1021/bc049880n CCC: $27.50 © 2004 American Chemical Society Published on Web 09/29/2004

Bis-Detergents for Macromolecular Drug Delivery

Bioconjugate Chem., Vol. 15, No. 6, 2004 1167

Figure 1. Two-step strategy for cytosolic delivery of macromolecules utilizing liposomes containing acid-labile bis-detergents such as BD2 in Scheme 1. The proposed two-step mechanism of action is as follows: In Step I, acid-catalyzed hydrolysis of the acetone acetal cross-linker in the bis-detergent within the endosome is expected to generate single-tailed detergents within the liposomal bilayer. Upon reaching a critical concentration, these tertiary amine detergents would promote liposomal collapse into micelles and triggered release of entrapped contents. In Step II, the detergent-containing micelles are expected to form stable or transient pores in the endosomal membrane, which in turn will facilitate cytosolic delivery of initially entrapped contents.

Figure 2. Size exclusion chromatography of BD1-containing liposomes encapsulating fluorescein-labeled, polyanionic dextran (MW 40 kDa) using a Sephadex CL-6B column. Rhodaminelabeled liposomes coelute with dextran in fractions no. 11 and 12 confirming entrapment. Later fractions (no. 22-25) contained unentrapped dextran.

ate carbonium ion by electron-donating groups (14, 15). In the current study, as an example of cleavable and noncleavable bis-detergents, we report the synthesis and characterization of BD1 and BD2, respectively (Figure 2). We demonstrate the ability of such agents to promote vesicle-to-micelle transition under acidic conditions and facilitate the endosome-to-cytosol translocation of macromolecules. EXPERIMENTAL PROCEDURES

Lipids and Chemicals. All chemicals and solvents were purchased from Aldrich (Milwaukee, WI) and used without further purification. Glass thin-layer chromatography (TLC) plates were also purchased from Aldrich. Egg phosphatidylcholine (PC), cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Triton X-100 and p-toluidinyl naphthalene sulfonic acid (TNS) was obtained from Sigma (St. Louis, MO). Fluorescein-labeled, polyanionic dextran of MW 40 kDa (Fl-Dex) was purchased from Molecular Probes (Eugene, OR). Antisense 18-mer oligonucleotides (5′-CCU CUU ACC UCA GUU ACA-3′, ON-705) with 2′-O-Me phosphorothioate back-

bones were prepared by The Midland Certified Reagent Company, Inc. (Midland, TX). An unrelated sequence of Texas red-labeled oligonucleotides (TR-ON) was a generous gift from Professor Ryszard Kole (Lineberger Cancer Center, UNC-CH). Synthesis of Bis-Detergents. The overall synthetic scheme is shown in Scheme 1. Compound 1 was synthesized by reacting (S)-glycidol with octadecylamine (16). Briefly, 3 g of octadecylamine (12 mmol) was dissolved using a heat gun in anhydrous methanol (5 mL) and transferred to a round-bottomed flask (round-bottom flask, 25 mL) fitted with a reflux condenser. A solution of (S)-glycidol (280 mg, 3 mmol) in 5 mL of methanol was then added dropwise to the round-bottom flask with continuous stirring and heat (60 °C). Reaction progress was monitored using TLC, and solvents were removed under vacuum after 1 h. The white residue was dissolved in hexane (50 mL) using a heat gun and allowed to cool, upon which a white precipitate (1) was formed. This process was repeated several times, following which the white precipitate was filtered and redissolved in 1 mL of CHCl3:MeOH:aq NH4OH (80:18:2). The clear solution was loaded onto a silica gel column (115 g) and purified using the same eluent system (Rf ∼ 0.4). Pure compound 1 was obtained at ∼60% yield (600 mg). Compound 2 was purchased from Aldrich, while compound 3 was prepared by the method of Lorette and Howard (17). Briefly, a mixture of 2-bromoethanol (0.6 mol, 75 g), 2,2-dimethoxypropane (0.25 mol, 26 g), benzene (100 mL), and p-toluenesulfonic acid (0.2 g) were added into a 500 mL round-bottom flask connected to a fractional distillation apparatus fitted with a vacuum pump. The reaction mixture was distilled until the benzene-methanol azeotrope (∼100 mL) boiling between 57 and 60 °C was completely removed. The contents were then cooled below the boiling temperature, and a solution of 0.5 g of sodium methoxide in 20 mL of methanol was added with stirring to achieve nearly instantaneous neutralization of acid. Distillation was resumed under vacuum (7 mmHg) upon which three fractions were collected: 28-30 °C (50 mL, fraction I); 65-75 °C (10 mL, fraction II); and 75-80 °C (2 mL, III). 1H NMR (CDCl3, 300 MHz): δ 1.39 (s, 6H), 3.45 (t, 4H), and 3.8 (t, 4H) confirmed pure symmetric acetone acetal (3) in fraction III. Bis-detergent BD1 was prepared by subjecting a mixture of compounds 1 (200 mg, 0.6 mmol), 2 (37 mg,

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Scheme 1. Synthetic Scheme for Preparation of Bis-Detergents BD1 and BD2

0.1 mmol), and Na2CO3 (100 mg) in 20 mL of CH3CN to reflux overnight. The solvents were then removed under high vacuum, and the residue was resuspended in CHCl3 and filtered using a sintered glass funnel. The concentrated filtrate was then loaded onto a silica gel column (115 g) and eluted using CHCl3:MeOH:aq NH4OH (80:18:2, Rf ∼ 0.6). Compound BD1 was obtained in good yield (65 mg, ∼80%). 1H NMR (CDCl3, 300 MHz, chemical shifts relative to TMS signal): δ 0.88 (t, 6H), 1.25 (br, 60H), 1.48 (br, 4H), 2.6-2.77 (m, 10H), 2.87-2.94 (m, 2H), 3.27 (br, 4H), and 3.49-3.84 (m, 14H), respectively. ESI-MS, calculated 801.76, found [M + Na]+ 824.7 (22%), 750.1 (72%), and 454.5 (6%). Bis-detergent BD2 was prepared in a similar fashion by subjecting a mixture of compounds 1 (335 mg, 1 mmol), 3 (70 mg, 0.2 mmol), and Na2CO3 (500 mg) in 20 mL of CH3CN to reflux overnight. A total of 72 mg (∼60% yield) of compound BD2 was obtained after silica gel chromatography using CHCl3:MeOH:aq NH4OH (80:18:2, Rf ∼ 0.6). 1H NMR (CDCl3, 300 MHz, chemical shifts relative to TMS signal): δ 0.88 (t, 6H), 1.25 (br, 60H), 1.38 (d, 6H), 1.45 (br, 4H), 2.5-2.8 (m, 10H), 2.85-2.95 (m, 2H), 3.45-3.53 (m, 10H), and 3.73.76 (m, 4H), respectively. ESI-MS, calculated 814.76, found [M + Na]+ 837.7 (12.5%), 763.7 (1%), 450.4 (37.5%), and 410.3 (49%). Preparation of Liposomes. Blank liposomes were prepared by generating a thin dry film composed of 7.5 mg of bis-detergent and 2.5 mg of PC in a 10 mL roundbottom flask by removing CHCl3 in vacuo. The film was hydrated using 1 mL of Tris-buffered saline (TBS, pH 8.0) or phosphate-buffered saline (PBS, pH 7.0) and alternatively vortexed and sonicated for 15 min. Vesicles were then extruded 10 times through a 0.22 µm polycarbonate membrane (Millipore) using an extruder (Lipex Biomembranes, Vancouver, CA). The size of vesicles formed was determined using a Nicomp 370 particle sizer (Particle Sizing Systems, CA). All macromolecules (fluorescently labeled or otherwise) were entrapped within liposomes using the minimum volume entrapment method (18). Briefly, a thin dry lipid

film containing BD1 or BD2 with PC (75:25), generated by removing chloroform under high vacuum, was hydrated with a 100 µL aliquot of TBS or PBS containing a high concentration of oligonucleotides (50 µM) or FlDex (10 µM), respectively, and vortexed at room temperature for 15 min. Vesicles formed were diluted to 1 mL with corresponding buffer and extruded (Lipex Biomembranes) 10 times through a 0.22 µm polycarbonate filter (Millipore) at a pressure of approximately 400 psi. Finally, unencapsulated solutes were separated from liposomal fractions by elution with TBS/PBS through Sepharose CL-6B (Sigma) or DEAE-Sepharose fast flow anion exchange (Pharmacia) resins packed in a 20 cm × 1 cm glass column. Liposomal fractions were identified by monitoring coelution of Rh-PE as a lipid marker and Fl-Dex (MW 40 kDa) as a fluid-phase marker, respectively. Encapsulation efficiency was determined by dissolving a 10 µL aliquot of liposomes in 100 µL of 2-propanol, diluting to 1 mL with PBS, and measuring the fluorescence intensity/absorbance of the sample. Final concentrations were calculated using the extinction coefficient of oligonucleotides or from a standard curve generated with known concentrations of Fl-Dex. Membrane-Bound pKa and Acid-Catalyzed Hydrolysis of Bis-Detergents. Membrane-bound pKa value of BD1 (10 mol %) incorporated into PC:cholesterol (70: 20) liposomes was determined using TNS (19). Acidcatalyzed hydrolysis of the acetone acetal cross-linker in BD2, which generates two, single-tailed lysosomotropic detergents and acetone (Scheme 2) was monitored by thin-layer chromatography. Briefly, 2 µL aliquots of BD2:PC (75:25) liposomes suspended in low ionic strength buffers of pH 4.0, 5.0, 6.0, and 7.0 were spotted at different time intervals onto silica gel TLC plates and eluted with CHCl3:MeOH:aq NH4OH (80:18:2). Spots corresponding to BD2 (Rf ∼ 0.6) and single-tailed, tertiary amine detergent monomers (Rf ∼ 0.3) were identified using ninhydrin. UV Light Scattering. Acid-induced collapse of BD1or BD2-containing liposomes into micelles was monitored for approximately 10 min using a Shimadzu Model



Scheme 2. Mechanism of Specific Acid-Catalyzed Hydrolysis of Bis-Detergent BD2 into Tertiary Amine Detergent Monomers and Acetone. The Formation of Stabilized Carbonium Ion Intermediate Is Thought To Be Rate-Determining (15)

UV2401-PC UV/Vis spectrophotometer. Briefly, a 30 µL aliquot of blank liposomes containing BD:PC (75:25) was suspended in 3 mL of Tris-buffered saline (pH 8.0) taken in a quartz cuvette and the decrease in absorbance (turbidity) monitored under different conditions at a wavelength of 350 nm. The buffer was acidified or alkalinized by quickly injecting a 10 µL aliquot of acetic acid or 10 µL aliquots of aq NH4OH into the cuvette. A 100 µL aliquot of 0.1% Triton X-100 injected at the end of the experiment served as positive control. Cytosolic Delivery of Antisense Oligonucleotides. For fluorescence microscopy experiments, HeLa Luc-705 cells obtained from Professor Ryszard Kole’s lab (Lineberger Cancer Center, UNC-CH) were cultured in two-well glass culture slides (2 × 105/well) using Dulbecco’s modified eagle’s medium (DMEM-H, Gibco), 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin under 5% CO2 humidified atmosphere at 37 °C. The cells were incubated for a period of 6 h with serum-free DMEM-H containing TR-ON (0.1 µM) in (a) free solution, (b) complexed with Lipofectamine (GIBCO, manufacturer specifications), (c) entrapped in BD1:PC (75:25) liposomes, or (d) entrapped in BD2:PC (75:25) liposomes. The formulations were replaced with serumcontaining media and incubated for a further 18 h after which the slides were washed thrice with cold phosphatebuffered saline (PBS) and fixed using 2% paraformaldehyde in PBS. Cells were then examined immediately under a Leica Diaplan fluorescence microscope (25× magnification) equipped with a Texas red filter and a Hamamatsu camera. For the antisense functional assay (20), HeLa Luc-705 cells were seeded in 12-well plates at 105 cells per well in DMEM-H containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin and allowed to adhere overnight in a 5% CO2 humidified atmosphere at 37 °C. The cells were incubated for a period of 6 h with serum-free DMEM-H containing ON705 (0.1 µM) in (a) free solution, (b) complexed with Lipofectamine (manufacturer specifications), (c) entrapped in BD1:PC (75:25) liposomes, or (d) entrapped in BD2:PC (75:25) liposomes. In a separate experiment, Hela Luc-705 cells were incubated with BD1:PC or BD2: PC liposomes containing ON-705 under similar conditions, but in serum-containing media to reduce apparent toxicity associated with such formulations. All formulations were then replaced with serum-containing media and incubated for a further 18 h after which the wells were washed thrice with Hank’s balanced salt solution (HBSS) without calcium/magnesium ions. An aliquot of 200 µL of cell lysis buffer (BD Pharmingen, San Diego, CA) was then added to each well, and

the cells were dislodged using a cell scraper (Costar, Corning, NY), following which the plates were incubated for 15 min at room temperature. The cell lysates were quickly spun down using a microcentrifuge to remove cell debris and a small aliquot used from each for determining protein content using the Bradford assay. Luminescence measurements were performed in a luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) using a Luciferase assay kit (BD Pharmingen, manufacturer specifications). Luciferase expression was expressed in relative light units (RLU) per µg of cellular protein. RESULTS

Entrapment of Macromolecules by Bis-DetergentContaining Liposomes. Liposomes containing BD1:PC: Rh-PE (75:24:1) were used to entrap Fl-Dex of MW 40 kDa in PBS (pH 7.0) using the minimum volume entrapment procedure. Following extrusion, liposomes and unentrapped solute were separated by size exclusion chromatography with PBS as eluent. The coelution of rhodamine-labeled liposomes (λex ) 530 nm; λem ) 590 nm) with Fl-Dex (λex ) 490 nm; λem ) 515 nm) in fractions no. 11 and no. 12 seen in Figure 2 indicates successful encapsulation. Un-entrapped Fl-Dex elutes in later fractions (no. 22-25). The average size of purified liposomes was 238 ( 69 nm with ∼40% encapsulation efficiency. Liposomes containing BD1 or BD2 with PC (75:25) entrapping oligonucleotides were prepared in a similar fashion and separated from unentrapped oligonucleotides by ion exchange chromatography. Encapsulation efficiencies of polyanionic oligonucleotides (∼6 kDa) and dextran (40 kDa) in BD-containing liposomes ranged from ∼10% in TBS to 40% in PBS. Membrane-Bound pKa of BD1 Incorporated into Liposomal Bilayers. The membrane-bound pKa of BD1 was determined using the TNS assay. Briefly, increasing membrane potential of BD1-containing liposomes with decreasing pH results in increased partitioning and intensity of TNS in liposomal bilayers. No pH-dependent change in membrane potential is seen with liposomes lacking BD1. As determined from Figure 3, the twin, tertiary amine headgroups have an average pKa of 6.37 ( 0.36. Acid-Catalyzed Hydrolysis of BD2 Embedded in Liposomes. To quickly estimate the sensitivity of the acetone acetal in BD2 toward acidic conditions relevant to an endosomal/lysosomal setting, a simple TLC assay was used. Briefly, ninhydrin was used to detect products of BD2 hydrolysis, i.e., detergent monomers upon incubation for different time intervals at 37 °C in buffers of


Figure 3. Membrane-bound pKa of BD1 determined using the TNS assay described previously. Liposomes containing PC/Chol with 10 mol % BD1 (b) and without BD1 (O) served as test and control, respectively. A pKa of 6.37 ( 0.36 was obtained from triplicate experiments with BD1-containing liposomes.

different pH. As shown in Figure 4, BD2 appears stable at 10 min in all buffers as indicated by the single spot at Rf ∼ 0.6. However, after an hour, detergent monomers are detectable at pH 4.0 and 5.0 (Rf ∼ 0.3), but not at pH 6.0 or 7.0. After 3 h, substantial hydrolysis of BD2 is seen in pH 4.0 buffer and about 50% of starting material is hydrolyzed at pH 5.0. Detergent monomers are also detectable at pH 6.0, but not at pH 7.0. Finally, after 6 h, the detergent dimers are almost completely hydrolyzed at pH 4.0, but still remain intact at pH 7.0. Thus, BD2 appears to degrade with a t1/2 of ∼3 h at pH 5.0 at 37 °C. Low pH-Induced Collapse of BD-Containing Liposomes into Micelles. Liposomal formulations containing 75 mol % of BD1 or BD2 were characterized at 37 °C for changes in turbidity by monitoring their UV absorption spectra at 350 nm. This wavelength is the region where changes in particle size will have a large effect on the optical density due to light scattering without any interference from lipid absorbance (20). UV spectra were obtained over a time interval of approximately 10 min during which liposomal suspensions in pH 8.0 buffer were (a) acidified to pH 4.2, (b) neutralized to pH 7.1, followed by addition of base increasing pH to 9.0, and (c) finally disrupted completely to micelles with 0.1% Triton X-100.

Asokan and Cho

As seen in Figure 5, addition of acetic acid to a final pH of 4.2 to liposomes containing BD1 (upper curve) results in ∼70% collapse into micelles. When the system was neutralized to pH 7.1, no changes in light scattering were apparent. In contrast, micellar aggregates were formed as suggested by the increase in turbidity observed at pH 9.0. The acidification of BD2-containing liposomal suspension (lower curve) resulted in nearly 90% collapse into micelles. Interestingly, neither neutralization nor alkalinization to pH 9.0 had an effect on turbidity. Addition of Triton X-100, which resulted in complete liposomal collapse into micelles served as reference. Cytosolic Delivery of Antisense Oligonucleotides. The intracellular disposition of Texas red-labeled oligonucleotides (TR-ON) was monitored using fluorescence microscopy (Figure 6). As seen in Panel 6C, liposomes containing acid-stable BD1 display large punctate spots against a partially diffuse background similar to that seen with Lipofectamine in Panel 6B. Such a distribution pattern suggests a moderate ability of BD1-containing liposomes to promote cytosolic delivery of oligonucleotides. In contrast, liposomes containing acid-labile BD2 show a largely diffuse pattern of fluorescence seen in Panel 6D. This observation is consistent with efficient endosomal permeabilization and cytosolic delivery of oligonucleotides by BD2-containing liposomes. Pinocytic uptake of free TR-ON, which is seen as a predominantly punctate pattern, served as control (Panel 6A). The ability of bis-detergent-containing liposomes to promote the cytosolic delivery of ON-705, an 18-mer oligonucleotide was also confirmed using an antisense functional assay based on luciferase up-regulation in HeLa Luc-705 cells (20). Briefly, the assay involves corrective splicing of aberrantly spliced luciferase precursor mRNA (pre-mRNA) to mRNA by ON-705 resulting in up-regulation of luciferase. It is noteworthy to mention that such corrective splicing strategies require backbonemodified antisense oligomers that function via RNase-H independent mechanism. Thus, oligomers with nucleaseresistant backbones, i.e., polyanionic 2′-O-methyl phosphorothioate (2′-O-Me) were used in this study. As seen in Figure 7, free ON-705 is unable to correct splicing and induce luciferase expression. On the other hand, as evidenced by similar levels of luciferase expression, liposomal formulations containing 75 mol % of BD1 or BD2 appear to be as efficient as Lipofectamine in promoting cytosolic delivery of oligonucleotides. In ad-

Figure 4. Acid-catalyzed hydrolysis of BD2, which generates single-tailed lysosomotropic detergents monitored by thin-layer chromatography. Briefly, 2 µL aliquots of BD2:PC (75:25) liposomes suspended in low ionic strength buffers of pH 4.0, 5.0, 6.0, and 7.0 were spotted at different time intervals onto silica gel TLC plates and eluted with CHCl3:MeOH:aq NH4OH (80:18:2). Spots corresponding to BD2 (Rf ∼ 0.6) and single-tailed, tertiary amine detergent monomers (Rf ∼ 0.3) were identified using ninhydrin.


Figure 5. Light scattering by BD1:PC (75:25) liposomes (upper curve) and BD2:PC (75:25) liposomes (lower curve) at 350 nm. Liposomal formulations were suspended in 3 mL Tris-buffered saline (pH 8.0) in a quartz cuvette and acidified to pH 4.2 with a 10 µL aliquot of acetic acid. Two 10 µL aliquots of aq NH4OH were added at ∼325 s and 375 s increasing the pH to 7.1 and 9.0, respectively. Finally, 100 µL of 0.1% Triton X-100 was used to disrupt vesicles completely (TX-100). All experiments were performed in duplicate.

dition, the ability of BD2-containing liposomes to deliver ON-705 to the cytosol and hence up-regulate luciferase expression appears to improve in the presence of serum in comparison with formulations containing the acidstable BD1. DISCUSSION

The bis-detergents characterized in this study were designed to overcome the issue of low levels of incorporation of single-tailed detergents in stable liposomal drug delivery systems. The strategy involves in situ generation of micelles within the endosome and subsequent cytosolic delivery of initially entrapped contents. Synthesis of the constructs is rather straightforward, although crosslinker 3 is often obtained in low yield. The acetone acetal linker in BD2 was chosen based on well-established chemistry and examples in the literature (17, 22). The kinetics of hydrolysis of BD2 incorporated in liposomes was assessed in a semiquantitative fashion using TLC. The acid-catalyzed hydrolysis of the acetone acetal spacer was rather sluggish (t1/2 ∼ 3 h; pH 5.0) as opposed to that reported in the literature (t1/2 ∼ 15 min; pH 5.0, (22)). Such a decrease in rate of hydrolysis can be attributed in part to the lower H3O+ activity at the bilayer-water interface in comparison to the bulk aqueous phase. In addition, the acquisition of cationic charge upon


protonation of tertiary amine headgroups of BD2 can further deplete H3O+ activity at the liposomal surface due to electrostatic charge repulsion (15, 23, 24). Indeed, the headgroups of bis-detergents in this study were shown to have an average pKa of 6.37, thus supporting the nearcomplete protonation of both tertiary amines at pH 5.0 or so. A detailed, quantitative analysis of the mechanism of acid-catalyzed hydrolysis of BD2 is currently in progress. Bis-detergents have been shown to form stable vesicles and more recently, been used to entrap macromolecules such as plasmid DNA (25). In the current study, lipid films containing 75 mol % detergent dimers and 25 mol % PC formed vesicles capable of entrapping macromolecules such as dextran. The higher entrapment efficiency in PBS can possibly be attributed to electrostatic interactions between polyanionic dextran and the partially protonated bis-detergent headgroups at neutral pH. This is further supported by the relatively lower encapsulation efficiency of formulations prepared at a higher pH in TBS (pH 8.0) when cationic charge on bis-detergent headgroups is negligible. We have observed that macromolecular probes such as dextran (MW 10kDa) can be delivered to the cytosol simply upon coincubation with micelles containing singletailed, pH-sensitive detergents (unpublished data). Such data in conjunction with other published results (5, 9) suggest that acid-induced collapse of liposomes into micelles within the endosome is a prerequisite for endosomal permeabilization and macromolecular escape into the cytosol. Results from the UV light scattering experiment shown in Figure 5, confirm the ability of bisdetergents to promote liposomal collapse into micelles under acidic conditions. Although this set of data does not allow interpretation of the observation at the molecular level, it is likely that the collapse was largely due to repulsion between the protonated twin headgroups of each bis-detergent. Such electrostatic repulsion between twin headgroups of a similar Gemini surfactant has been shown to promote lamellar-to-micellar transition (25). However, such low pH-induced vesicle-to-micelle transition appears to be reversible, presumably due to reaggregation of micelles at basic pH (26). In contrast, the acidtriggered collapse of liposomes containing acid-labile BD2 appears irreversible. In summary, it is tempting to speculate that the irreversible, acid-catalyzed hydrolysis of BD2 into two single-tailed detergents might well be responsible for such efficient liposomal collapse and micelle formation. Pilot studies were therefore conducted to investigate the hypothesis that BD2 could promote endosomal escape more efficiently than its noncleavable counterpart, BD1.

Figure 6. Intracellular distribution of different formulations containing Texas red-labeled oligonucleotides (TR-ON). HeLa Luc705 cells grown in slide wells were incubated for a period of 6 h with serum-free DMEM-H containing TR-ON (0.1 µM) in (A) solution, (B) complexed with Lipofectamine (GIBCO, manufacturer specifications), (C) entrapped in BD1:PC (75:25) liposomes, or (D) entrapped in BD2:PC (75:25) liposomes. Formulations were replaced with serum-containing media and cells incubated for 18 h after which slides were washed thrice with cold PBS and fixed using 2% paraformaldehyde in PBS. Slides were immediately examined under a Leica Diaplan fluorescence microscope (25× magnification) equipped with a Hamamatsu camera.


Figure 7. Upregulation of luciferase upon cytosolic delivery of antisense oligonucleotides (ON-705) using different formulations. HeLa Luc-705 cells seeded in 12-well plates were treated for 6 h with serum-free DMEM-H containing ON-705 (0.1 µM) in: solution (n ) 6); complexed with Lipofectamine (n ) 6, manufacturer specifications); entrapped in BD1:PC (75:25) liposomes (n ) 3); entrapped in BD2:PC (75:25) liposomes (n ) 3); or in serum-containing DMEM-H with BD1:PC or BD2:PC liposomes (n ) 3 each). Formulations were replaced with serumcontaining media and luciferase expression measured after 24 h in a luminometer using a Luciferase assay kit (manufacturer specifications). Luciferase expression is expressed in relative light units (RLU) per µg of cellular protein (mean ( SD, p < 0.05).

The cytosolic delivery potential of bis-detergent formulations was assessed qualitatively by monitoring the intracellular distribution of initially entrapped TR-ON through fluorescence microscopy. The predominantly diffuse pattern seen in HeLa cells treated with formulations containing acid-labile BD2 contrasts the partially aggregated fluorescence pattern seen in cells treated with BD1-containing liposomes or Lipofectamine. These results are consistent with the notion that acid-labile bisdetergents are likely more efficient in promoting endosome-to-cytosol translocation of macromolecules than noncleavable detergent dimers or cationic lipids. The delivery efficiency of aforementioned formulations was quantitatively assessed using an antisense functional assay. In the absence of serum, formulations of ON-705 prepared with bis-detergents were as efficient as Lipofectamine in promoting luciferase upregulation. Interestingly, addition of serum, which is known to completely inhibit Lipofectamine-mediated transfection (27, 28) did not attenuate cytosolic delivery potential of BD1- or BD2containing liposomes. More importantly, liposomes containing acid-labile BD2 are presumably more efficient at promoting cytosolic delivery of ON-705 than liposomes containing acid-stable BD1 in the presence of serum. In summary, acid-labile bis-detergents such as BD2 appear to offer a dual advantage, i.e., the formulation of stable liposomes at neutral pH as well as in situ generation of membrane-disrupting, single-tailed detergents that promote efficient liposomal collapse into micelles in acidic environments. Such a system can potentially be exploited for cytosolic delivery of macromolecular drugs such as proteins, oligonucleotides, and genes in vitro and in vivo. CONCLUSION

Two detergent dimer constructs, also referred to as ‘bisdetergents’ in the present study, with noncleavable (BD1) or acid-labile (BD2) spacers were synthesized. The bisdetergents were characterized for their ability to form stable liposomes at concentrations as high as 75 mol %,

Asokan and Cho

for the pH-dependent protonation of their headgroups and the ability of BD2, in particular to hydrolyze under acidic conditions. Liposomes composed of 75% BD1 or BD2 and 25% PC collapsed into micelles at mildly acidic pH similar to that found in endosomes/lysosomes. The collapse of BD2:PC liposomes was more efficient than that of BD1:PC liposomes presumably due to acidcatalyzed hydrolysis of the acetal spacer. Such can be attributed to the accumulation of single-tailed, micellefavoring detergent species within the liposomal bilayer. Bis-detergent-containing liposomes were utilized to encapsulate antisense oligonucleotides and promote delivery of entrapped contents into the cytosol of HeLa cells. The efficiency of cytosolic delivery was comparable to Lipofectamine and substantially improved in the presence of serum. Further improvements in the cytosolic delivery potential of acid-sensitive bis-detergents can possibly be achieved by incorporation of biodegradable linkers and systematic characterization of formulations thereof. These studies are currently in progress. ACKNOWLEDGMENT

We would like to thank Professors Rudy Juliano, Hal Kohn, Jeff Krise, Barry Lentz, and Ed LeCluyse for their helpful suggestions on this study. We would also like to acknowledge NIH GM071040 for research support. LITERATURE CITED (1) Cullis, P. R., and De Kruijff, B. (1978) The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study. Biochim Biophys Acta 513 (1), 31-42. (2) Tari, A., and Huang, L. (1989) Structure and function relationship of phosphatidylglycerol in the stabilization of the phosphatidylethanolamine bilayer. Biochemistry 28 (19), 7708-7712. (3) Winterhalter, M., and Lasic, D, D. (1993) Liposome stability and formation: experimental parameters and theories on the size distribution. Chem. Phys. Lipids 64 (1-3), 35-43. (4) Guo, X., and Szoka, F. C. (2001) Steric stabilization of fusogenic liposomes by a low-pH sensitive PEG- -diortho esterslipid conjugate. Bioconjugate Chem. 12 (2), 291-300. (5) Asokan, A., and Cho, M. J. (2003) Cytosolic delivery of macromolecules. II. Mechanistic studies with pH-sensitive morpholine lipids. Biochim. Biophys. Acta 1611 (1-2), 151160. (6) Bhamidipati, S. P., and Hamilton, J.A (1995) Interactions of lyso 1-palmitoylphosphatidylcholine with phospholipids: a 13C and 31P NMR study. Biochemistry 34 (16), 5666-5677. (7) Sun, C., Hanasaka, A., Kashiwagi, H., and Ueno, M. (2000) Formation and characterization of phosphatidylethanolamine/ lysophosphatidylcholine mixed vesicles. Biochim. Biophys. Acta 1467 (1), 18-26. (8) Liang, E., and Hughes, J. A. (1998) Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166 (1), 37-49. (9) Asokan, A., and Cho, M. J. (2004) Cytosolic delivery of macromolecules: IV. Mechanism of membrane destabilization by pH-sensitive detergents. Biochim. Biophys. Acta (under review). (10) Boomer, J. A., and Thompson, D. H. (1999) Synthesis of acid-labile diplasmenyl lipids for drug and gene delivery applications. Chem. Phys. Lipids 99 (2), 145-153. (11) Jaeger, D. A., Li, B., and Clark, T. (1996) Cleavable doublechain surfactants with one cationic and one anionic headgroup that form vesicles. Langmuir 12 (18), 4314-4316. (12) Yaroslavov, A. A., Udalykh, O., Melik-Nubarov, N. S., Kabanov, V. A., Ermakov, Y. A., Azov, A. V., and Menger, F. M. (2001) Conventional and gemini surfactants embedded within bilayer membranes: contrasting behavior. Chem.Eur. J. 7 (22), 4835-4843.


Bis-Detergents for Macromolecular Drug Delivery (13) Menger, F. M., and Peresypkin, A. V. (2003) Strings of vesicles: flow behavior in an unusual type of aqueous gel. J. Am. Chem. Soc. 125 (18), 5340-5345. (14) Fife, T. H., and Jao, L. K. (1965) Substituent effects in acetal hydrolysis. J. Org. Chem. 30 (5), 1492-1495. (15) Cordes, E. H., and Bull, H. G. (1974) Mechanism and catalysis for hydrolysis of acetals, acetals and orthoesters. Chem Rev. 74 (5), 581-603. (16) Vollhardt, D., and Gehlert, U. (2002) Chiral discrimination in 1-stearylamine-glycerol monolayers. J. Phys. Chem. B. 106 (17), 4419-4423. (17) Lorette, N. B., and Howard, W. L. (1960) Preparation of ketals from 2,2-dimethoxypropane. J. Org. Chem. 25 (4), 521525. (18) Thierry, A. R., Rahman, A., and Dritschilo, A. (1993) Overcoming multidrug resistance in human tumor cells using free and liposomally encapsulated antisense oligodeoxynucleotides. Biochem. Biophys. Res. Commun. 190 (3), 952-960. (19) Bailey, A. L., and Cullis, P. R. (1994) Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids. Biochemistry 33 (42), 12573-12580. (20) Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37 (18), 6235-6239. (21) Ollivon, M., Eidelman, O., Blumenthal, R., and Walter, A. (1988) Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside. Biochemistry 27 (5), 1695-1703.

(22) Srinivasachar, K., and Neville, D. M., Jr. (1989) New protein cross-linking reagents that are cleaved by mild acid. Biochemistry 28 (6), 2501-2509. (23) Adamson, A. W., and Gast, A. P. (1997) Electrical aspects of surface chemistry. Physical chemistry of surfaces, 6th ed., pp 169-179, John Wiley & Sons, New York. (24) Guo, X., MacKay, J. A., and Szoka, F. C., Jr. (2003) Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester poly(ethylene glycol) lipid. Biophys. J. 84 (3), 1784-1795. (25) Fielden, M. L., Perrin, C., Kremer, A., Bergsma, M., Stuart, M. C., Camilleri, P., and Engberts, J. B. (2001) Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro. Eur. J. Biochem. 268 (5), 1269-1279. (26) Johnsson, M., Wagenaar, A., and Engberts, J. B. (2003) Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH. J. Am. Chem. Soc. 125 (3), 757-760. (27) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Soc. 84 (21), 7413-7417. (28) Yang, J. P., and Huang, L. (1997) Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther. 4 (9), 950-960.

BC049880N

Cytosolic Delivery of Macromolecules. 3. Synthesis and

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