Endosomolysis by Masking of a Membrane-Active Agent - American

Department of Pediatrics and Medical Genetics, Medical School, University of WisconsinsMadison,. Madison, Wisconsin 53705. Received August 15, 2002; ...
0 downloads 0 Views 200KB Size
Download PDF
Bioconjugate Chem. 2003, 14, 51−57

51

Endosomolysis by Masking of a Membrane-Active Agent (EMMA) for Cytoplasmic Release of Macromolecules David B. Rozema,*,† Kirk Ekena,† David L. Lewis,† Aaron G. Loomis,† and Jon A.Wolff‡ Mirus Corporation, 505 S. Rosa Road, Madison, Wisconsin 53711, and Waisman Center, Department of Pediatrics and Medical Genetics, Medical School, University of WisconsinsMadison, Madison, Wisconsin 53705. Received August 15, 2002; Revised Manuscript Received November 6, 2002

Endosomolysis, a critical barrier to efficient delivery of macromolecules such as nucleic acids, has been breached using a novel approach: endosomolysis by masking of a membrane-active agent (EMMA). To demonstrate the concept of EMMA, a cationic membrane-active peptide, melittin, was reversibly inhibited using a maleic anhydride derivative. At neutral pH, the lysines of melittin are covalently acylated with the anhydride, thereby inhibiting melittin’s membrane disruption activity. Under acidic conditions such as those present within endosomes, the amide bond of the maleamate is cleaved, thus unmasking melittin. The active melittin can then disrupt the endosomal membrane resulting in release of biologically active molecules into the cytoplasm. This approach avoids cellular toxicity by restricting melittin’s activity until it reaches the endosomal compartment. The utility of this approach was demonstrated by delivery phosphorodiamidate morpholino oligonucleotides (PMOs).

INTRODUCTION

In the rational design of synthetic delivery vehicles for biologically active compounds and macromolecules such as nucleic acids, endosomal escape can be a critical barrier for their efficient delivery to cytoplasmic and nuclear sites of action (1-3). Endosomal escape can be enabled by the use of liposomes that hypothetically fuse with the endosomal membrane in a process akin to that used by enveloped viruses such as influenza (4, 5). Alternatively, nonliposomal vehicles (e.g., polymer-based systems) and nonenveloped viruses (e.g., adenoviruses) must escape from endosomes by disrupting their membranes (termed endosomolysis) (6). Despite some advances, our understanding of endosomal release and ability to synthetically enhance it remain at a rudimentary level. Both viral and synthetic processes for accomplishing endosomal release often rely upon the changing environment of the endosome and/or lysosome to trigger either membrane fusion or disruption (7, 8). Endosomal acidification is frequently exploited to facilitate escape, given that as the endosome matures into a lysosome, the pH progressively drops to less than 5 (9-11). The pH gradient between cytoplasm and endosome causes endosomolytic monoamines such as chloroquine to concentrate within endosomes and destroy the pH gradient (12). The pH-sensitive amines within polyamines such as polyethyleneimine also play a role in endosomal release (1315). For some liposome-based systems, pH-sensitive groups, typically carboxylic or amino groups, have been incorporated into lipids that undergo phase transitions upon protonation (4, 16). Peptides and synthetic polymers containing protonable groups have been modeled after viral sequences to become more amphipathic and membrane active in acidic environments (17-19). By limiting membrane activity to acidic vesicles, effects on the * Corresponding author. E-mail: [email protected]. † Mirus Corporation. ‡ University of WisconsinsMadison.

plasma membrane and thereby cellular toxicity are attenuated. The use of protonation to effect membrane disruption is beset with a potential conundrum. Endosome disruption by a pH-sensitive agent will destroy the pH gradient, which may reverse the membrane activity of the endosomolytic agent. As a consequence, the endosome membrane could reseal before the macromolecule diffuses out of the endosome, thereby limiting delivery. To avoid the swift reversibility of the protonation-based membrane active agents, we have developed an irreversible process that relies upon chemical bond cleavage to unmask a peptide’s endosomolytic activity. The use of linkages that are labile within the endosomal or lysosomal milieu has previously been used in liposomes or for coupling drugs with carriers (4, 7, 20, 21). We chose to work with the cationic peptide melittin that contains several lysines required for its membrane activity and to use maleic anhydrides to reversibly modify the  amino groups of the lysine residues. Maleic anhydrides react with amines to form pH-labile amides, called maleamic acids, that are cleaved when the carboxylic group becomes protonated (22). A series of cyclic, maleic anhydride derivatives were evaluated for their ability to reversibly inhibit melittin (Figure 1A). Citraconic anhydride had previously been used to reversibly modify the primary amine of dioleoylphosphatidylethanolamine, but this reagent was not effective for our purposes (21). A previously unstudied maleic anhydride derivative, 2-propionic-3-methylmaleic anhydride (termed CDM for carboxylated dimethyl maleic acid), was synthesized to incorporate two design features that were found to be required: disubstitution of the maleic anhydride to increase pH-lability and an additional carboxylic acid to inhibit membrane activity of the peptide. MATERIALS AND METHODS

Maleic Anhydride Derivatives and Synthesis of 2-Propionic-3-methylmaleic Anhydride (Carboxydimethylmaleic Anhydride or CDM). 2-Propionic-3-

10.1021/bc0255945 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/19/2002

52 Bioconjugate Chem., Vol. 14, No. 1, 2003

Rozema et al.

Figure 1. (A) Maleic anhydride and maleamate derivatives: maleic R1 and R2dH, dimethyl maleic R1 and R2 ) CH3, citraconic R1or2 ) H and R2or1 ) CH3, cis-aconitic R1or2 ) H and R2or1 ) CH2CO2H, and 2-propionic-3-methylmaleic (CDM) R1or2 ) CH3 and R2or1 ) C2H4CO2H. (B) Synthesis of 2-propionic-3-methylmaleic anhydride (CDM): a Horner-Emmons reaction between dimethyloxoglutarate and triethyl-2-phosphonopropionate, followed by saponification of the ester groups.

methylmaleic anhydride was synthesized according to a published procedure (23). To a suspension of sodium hydride (0.58 g, 25 mmol) in 50 mL anhydrous tetrahydrofuran was added triethyl-2-phosphonopropionate (7.1 g, 30 mmol). After evolution of hydrogen gas had stopped, dimethyl-2-oxoglutarate (3.5 g, 20 mmol) in 10 mL anhydrous tetrahydrofuran was added and stirred for 30 min. Water, 10 mL, was then added, and the tetrahydrofuran was removed by rotary evaporation. The resulting solid and water mixture was extracted with 3 × 50 mL ethyl ether. The ether extractions were combined, dried with magnesium sulfate, and concentrated to a light yellow oil. The oil was purified by silica gel chromatography elution with 2:1 ether:hexane to yield 4 g (82% yield) of pure triester. The 2-propionic-3-methylmaleic anhydride was then formed by dissolving of this triester into 50 mL of a 50/50 mixture of water and ethanol containing 4.5 g (5 equiv) of potassium hydroxide. This solution was heated to reflux for 1 h. The ethanol was then removed by rotary evaporation, and the solution was acidified to pH 2 with hydrochloric acid. This aqueous solution was then extracted with 200 mL ethyl acetate, which was isolated, dried with magnesium sulfate, and concentrated to a white solid. This solid was then recrystallized from dichloromethane and hexane to yield 2 g (80% yield) of 2-propionic-3-methylmaleic anhydride (CDM). Citraconic, cis-aconitic, dimethylmaleic, and succinic anhydrides were purchased from Aldrich. Quantification of Amines by TNBS. To determine the amine content of a sample, 50-200 nmol of amine is added to a 0.5 mL solution of 100 mM Borax solution containing 0.4 mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma). Fifteen minutes later the absorbance of the solution was measured at 420 nm. The amount of amine may be calculated by the absorbance of a solution of known amine concentration. Acylation of Poly-L-lysine (PLL) with CDM, Dimethylmaleic, Succinic, Citraconic, and cis-Aconitic Anhydrides and Assessment of Charge Density. To a solution containing 200 µg PLL (MW 34 000 from Aldrich), 2 mg HEPES, and 0.4 mg NaOH in 100 µL water was added 0.4 mg of CDM, dimethylmaleic, suc-

cinic, or citraconic anhydride in 20 µL ethanol with rapid vortexing. For cis-aconitic anhydride, there was a substantial amount of hydrolyzed anhydride present in the sample; therefore, 2 mg of anhydride, 12 mg of HEPES, and 2.4 mg of NaOH were used. By TNBS assay there was no detectable amount of amine upon acylation under these conditions. The charge density of these polyanions was determined by adapting a technique used for assessing DNA condensation (24, 25). A compacted, interpolyelectrolyte complex is formed when the polyanions (PLL completely reacted with the anhydrides) are mixed with fluoresceinlabeled PLL. The compaction causes the fluorescein residues on PLL to be in closer proximity and self-quench, which enables the condensation state and by inference the effective charge density of the polyanions to be conveniently determined. To measure the condensation of PLL by acylated PLL, 10 µg of fluorescein-labeled PLL was placed in 0.5 mL of 5 mM HEPES buffer at pH 7.8. The fluorescence intensity of this solution was measured (excitation at 495 nm, emission at 530 nm). A 1 µg sample of acylated PLL (weight based on the starting weight of PLL, not on the weight of acylated PLL) was added to the fluoroscein-labeled PLL, and the fluorescence intensity was again measured. This was repeated until the decrease in fluorescence intensity ceased. The amount of acylated PLL needed to quench fluorescein-PLL to the maximum degree was assumed to be point at which an equal amount of polyanion was added to the polycation. Acylation of Melittin with CDM, dImethylmaleic, Citraconic, and cis-Aconitic Anhydrides. To a solution containing 200 µg melittin, 500 µg HEPES, and 100 µg NaOH in 20 µL water was added 100 µg of CDM, dimethylmaleic, or citraconic anhydride in 50 µL ethanol with rapid vortexing. For cis-aconitic anhydride 250 µg of anhydride, 1.25 mg of HEPES, and 250 mg of NaOH were used. By TNBS assay there was no detectable amount of amine upon acylation under these conditions. Fluorescein Labeling of PLL. To a solution of PLL (10 mg) in 1 mL 10 mM K2CO3 was added 0.4 mg of fluorescein isothiocyanate (0.02 functional eq). After 2 h, the polymer was placed into dialysis tubing (12 000 MW cutoff) and dialyzed for 72 h against 3 × 2 L deionized

Endosomolysis by Masking of a Membrane-Active Agent

water. The polymer was then removed from the tubing, and the water was removed by lyophilization. The yield of the reaction was estimated to be 50% by measurement of the absorbance of flourescein-labeled PLL at 494 nm in a solution of 10 mM NaHCO3 using the extinction coefficient 77 000 M-1cm-1(Molecular Probes). Synthesis of Fluorescein-PEG. A 100 mg sample of monoamine PEG (MW 5000, Shearwater) was dissolved 1 mL of dimethylformamide. To this solution was added 3.5 µL of diisopropylethylamine and 8 mg of fluorescein isothiocyanate. After 1 h, the PEG was precipitated by addition of 10 mL of diethyl ether. The PEG was then dissolved in 5 mL of water and purified by size exclusion chromatography using a 30 × 150 mm column of sephadex G10 (Sigma) eluting with water. Kinetics of Acid-Catalyzed Cleavage of Maleamylated Glycinylalanine. CDM, dimethyl maleic, and citraconic modified glycinylalanine (GA, from Sigma) were synthesized by addition of 400 µg of CDM, citraconic, or dimethylmaleic anhydride in 20 µL of ethanol to a solution of 200 µg of GA and 2.4 mg of HEPES base in 44 µL of water. The solutions were mixed by rapid vortexing. The acylated peptide was acidified by addition of 0.8 µL glacial acetic acid and 10 µL of 1 M sodium acetate to bring the pH to 5.0. At various times, 10 µg aliquots were removed and added to 0.5 mL of 100 mM NaHCO3 solution containing 0.4 mM TNBS. Fifteen minutes later the absorbance of the solution was measured at 420 nm. A plot of ln [1 - (At/A0)] as a function of time was a straight line whose slope is -k, the rate constant for the cleavage reaction, where At is the absorbance at time t and A0 is the absorbance of unmodified GA. For cis-aconitic modification of GA, 3-fold more anhydride, base, and acid were used to synthesize and cleave the acylated peptide. Hemolysis Assay. The membrane activity of polymers and peptides was measured using a red blood cell (RBC) hemolysis assay (26). Porcine whole blood was isolated in heparin-containing vacutainers. The RBC's were isolated by centrifugation at 500 RCF for 5 min. They were washed three times with 100 mM dibasic sodium phosphate at the desired pH and resuspended to the initial volume. The desired pH phosphate buffer was obtained by acidification of a dibasic sodium phosphate stock with HCl. (Buffers made from mixing of mono- and dibasic phosphate lysed red blood cells at acidic pH.) A 20 µL sample of the washed RBC suspension, which is approximately 108 cells (26), was added to 500 µL of phosphate buffer. To this solution was added various amounts of peptide or polymer. The samples were incubated for 1.5 h in a 37 °C incubator. They were then spun for 1 min at 15 000 RCF. Lysis was determined by measuring the absorbance of the supernatant at 541 nm. Percent hemolysis was calculated assuming 100% lysis to be the absorbance of hemoglobin released upon addition of deionized water; all sample absorbances had the absorbance of buffer alone subtracted. Fluorescein-PEG Delivery Assay. HeLa (human epitheloid) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro, Herndon, VA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah) in a humidified incubator at 37°C with 5% CO2 atmosphere. Cells were plated in 6-well culture dishes containing untreated glass coverslips at a density of 1.01.5 × 105 cells/well and incubated for 24-48 h. Media were replaced with 1.0 mL DMEM containing 1.0 mg fluorescein-PEG3000 either with or without 400 µg CDM-melittin. Cells were then incubated for 10 min at

Bioconjugate Chem., Vol. 14, No. 1, 2003 53

37 °C, washed once with warm 37 °C DMEM, and incubated for an additional 35 min in DMEM containing 10% bovine serum. Cells were then washed three times with PBS (Sigma), fixed for 30 min at 4 °C in PBS + 4% formaldehyde (Sigma), and washed three times in PBS. Coverslips were mounted onto glass slides for fluorescent microscopy. Images of the samples were collected by confocal microscopy on a Zeiss LSM510 confocal microscope (Zeiss, Germany) using a 63× oil plan apo objective with NA 1.4. Similar methods were used with 1 mg/mL of fluorescein-dextran (10 kDa, purchased from Molecular Probes). To assay or cellular toxicity, 2 µL of saturated solution propidium iodide in water was added to cells in 3 mL PBS after 35 min incubation in DMEM. After 3 min, the PBS solution was removed and cells were washed and fixed as described above. To study the effect of a proton pump inhibitor on the release of fluorescein-PEG, the cells were incubated with 200 nM bafilomycin A, while the cells were loaded with flourescein-PEG and during their incubation in the presence of DMEM. The cells were then washed and fixed as described above. No diffuse cytoplasmic staining was observed for cells incubated with bafilomycin A. Oligonucleotide Delivery Assay. HeLa Luc/705 cells (Gene Tools, Philomath OR) were grown under conditions used for HeLa cells. The cells were plated in 24-well culture dishes at a density of 3 × 106 cells/well and incubated for 24 h. Media were then replaced with 1.0 mL DMEM or 1.0 mL DMEM containing 10% FBS. To these samples was added 2.5 nmol PMO (CCT CTT ACC TCA GTT ACA ATT TAT A, Gene Tools, Philomath, OR) with or without 2.5 or 5.0 nmol CDM-melittin, citraconylated melittin, or cis-aconitylated melittin. The cells were incubated for 4 h in a humidified, 5% CO2 incubator at 37°C. The medium was then replaced with DMEM containing10% fetal bovine serum. The cells were then incubated for an additional 48 h. The cells were then harvested and the lysates were then assayed for luciferase expression as previously reported using a Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer (27). RESULTS AND DISCUSSION

Synthesis of a carboxylic acid-containing derivative of dimethylmaleic anhydride(CDM). Two features of maleic anhydride derivatives were systematically evaluated. The first was the degree of substitution at the unsaturated carbon-carbon bond since it has been shown that the greater amount of substitution increases pH-lability (28, 29). The parent anhydride, maleic anhydride, has no substitution and forms the most stable maleamic acid (Figure 1). Citraconic and cis-aconitic-derived maleamic acids have one substitution and are more pH-labile. Maleamic acids derived from disubstituted dimethyl maleic anhydride are the most pH-labile (30). The second parameter evaluated was the effect of the addition of a carboxylate-containing group, which increases charge and water solubility. The cis-aconitic anhydride has such a carboxylic acid group. To complete the series and have a disubstituted anhydride with an additional carboxylic acid group, CDM was synthesized via a Horner-Emmons reaction between dimethyloxoglutarate and triethyl-2phosphonopropionate, followed by saponification of the ester groups (Figure 1B) (23). Acylation of a Polyamine with Cyclic Anhydrides and the Effect on Charge Density. To confirm the ability of the anhydrides to modify a polyamine and to

54 Bioconjugate Chem., Vol. 14, No. 1, 2003

Rozema et al.

Figure 2. The quenching of fluorescein-labeled PLL upon addition of PLL modified with succinic anhydride (3),dimethylmaleic anhydride (b), CDM anhydride (O), citraconic (9), or cis-aconitic anhydride (0).

Figure 3. The cleavage of maleamic acid modifications of the glycinylalanine dipeptide modified with dimethylmaleic (b), CDM (O), citraconic (9), or cis-aconitic anhydride (0).

evaluate the effect of acylation on the charge of the amines, the cyclic anhydrides were reacted with the polyamine PLL. The effect of acylation on the charge of the modified polyamine is important, given that the membrane activity of melittin is presumably dependent on its positive charge (vide infra). After reaction of PLL with succinic, citraconic, cis-aconitic, dimethylmaleic, and CDM anhydrides, the TNBS assay indicated that there was complete conversion of the γ-amines of PLL to carboxylates. The charge density of these polyanions was determined by assessing their ability to condense cationic, fluorescein-labeled PLL (24). At slightly basic pH, 1-1.2 functional equiv of succinylated PLL is required to condense PLL, which is estimated from our observation that 10-12 µg of succinylated PLL is required to fully quench 10 µg of fluorescein-labeled PLL (Figure 2). Similarly, 1-1.3 equiv of citraconylated PLL and 1.5 equiv of cis-aconitylated PLL, which has two carboxylate groups per repeating unit, are required to condense PLL. In contrast, PLL that is modified with dimethylmaleic anhydride requires ca. 20 equiv to condense PLL. Similarly, the charge density of CDM-modified PLL is roughly one charge per two carboxylates. It appears that the distal carboxylate of CDM anhydride adds charge to the modified polymer while the carboxylate of the anhydride contributes very little charge to the polyanion. This deficiency of effective charge density for dimethylmaleamates may be due to a hydrogen bond interaction between the amide proton and the carboxylate of the dimethymaleamylate group. Another hypothesis is that the steric bulk of the two methyl groups prevents any interaction. The reported pKa of the carboxylate of dimethymaleamylate is 4.2 (28), and the pKa of citraconylates is 3.2. These values suggest that the carboxylate is not hydrogen bonded to the amide proton because one would expect that a hydrogen bond between the carboxylate and amide proton would decrease, not increase the pKa of dimethymaleamylate. Whatever the reason for dimethylmaleamate’s apparent lack of charge density, these results indicate that CDM can modify the amines of lysine, thereby converting a positive charge into a negative one. The Acylation of the Dipeptide Glycinylalanine and the Kinetics of Cleavage. The rates of acidcatalyzed cleavage of the maleamates were evaluated using the dipeptide glycinylalanine (GA) (Figure 3). GA was chosen because it (1) contains one amine group, which simplifies kinetics, and (2) contains a carboxylate distant from the amine, which keeps the peptide soluble in the assay conditions without greatly affecting the pKa of the amine. The rate of maleamate cleavage was studied

by addition of the modified peptides to a pH 5 solution, and at various times the cleavage reaction was quenched by addition to a pH 9 solution containing TNBS. As expected, the disubstituted maleamic acids cleave much more rapidly than monosubstituted maleamic acids (Figure 3) (29). At early time points, the reverse reaction is negligible, and the reaction can be treated as unimolecular; but at later time points, the reverse reaction between amine and anhydride affects the concentration of amine, and the reaction is no longer strictly unimolecular. Data points where the reaction is less than 50% complete can be fitted to the equation ln[1 - (At/A0)] ) -kt, where At is the absorbance at time t and A0 is the absorbance of PLL that was not modified by anhydride. We calculated rate constants of 0.4 min-1 for dimethylmaleamic acid cleavage and 0.3 min-1 for the cleavage of CDM modified GA, which correspond to half-lives of 1.5 and 2 min, respectively. Similar measurements of the reversal of citraconic and cis-aconitic modification revealed much slower cleavage kinetics with an approximate half-life of 300 min. Acylation of the Membrane Disruptive Peptide Melittin. To investigate the effect of acylation on the activity of membrane-active, cationic peptides, we chose melittin. Melittin is a 26-residue peptide from bee venom (GIGAILKVLATGLPTLISWIKNKRKQ from the little honey bee), which is highly cytotoxic and hemolytic (31). Many synthetic analogues of melittin have been studied to determine which residues are important for membrane disruptive ability (17). The lysine at position 7 has been shown to be important for hemolytic activity (32). This importance of lysine 7 and the presence of four other amine groups, from three lysines and the amino terminus, suggest that regulating these amino groups could control the activity of melittin. To test this hypothesis, we acylated melittin at pH 7.5 with two molar equiv, relative to the four lysine residues with succinic, cis-aconitic, dimethylmaleic, citraconic, and CDM anhydrides. Modification by all of the anhydrides except dimethylmaleic anhydride resulted in a complete loss of membrane activity as measured by red blood cells lysis. Measurement of amine content by TNBS revealed that the inactivation of melittin by dimethylmaleic anhydride modification was not the result of incomplete amine acylation. A possible explanation for the activity of dimethylmaleamylated melittin is the lack of effective charge for dimethymaleamates. As we observed that dimethylmaleamylated PLL possessed very little charge density, we would expect that dimethylmaleamylated melittin would not be entirely inhibited from interacting with the anionic cellular membrane.

Endosomolysis by Masking of a Membrane-Active Agent

Figure 4. The return of melittin hemolytic activity at various times after incubation of maleamic-modified melittin at pH 5.0, for dimethylmaleic- (b), CDM- (O), citraconic- (9), or cis-aconiticanhydride- (0) modified melittin.

Restoration of Membrane Activity of Melittin upon Acidification. To test whether the reversibility of the acylation reaction restores membrane activity, melittin modified with CDM, citraconic, or cis-aconitic anhydride was incubated at pH 5. At various times, the acidification was stopped by addition to pH 7.5 phosphate buffer and the membrane disruptive ability of each sample was measured by the addition of red blood cells. The membrane activity for CDM-melittin returned to 100% within 25 min (Figure 4). Analysis of the kinetics of the return of activity using an analysis method similar to that used for the TNBS assay (plotting of ln[1 - (At/ A0)] as a function of time to determine the slope, -k) revealed a rate constant of 0.07 min-1, or a half-life of 10 min for CDM-melittin. The half-life for the restoration of melittin’s activity is roughly 4 times longer than the half-life of cleavage. This increase suggests that most, if not all, of the CDM groups must be cleaved from melittin for it to be active. Despite this delay for the return of activity, these results suggest that the new reagent CDM is able to reversibly inhibit melittin’s membrane activity in a time frame consistent with endocytosed materials’ transit to late endosomes (11). As one would expect from the kinetics for the cleavage of monosubstituted maleamic acids, the cleavage of citraconic and cis-aconitic anhydride modifications of melittin were much slower (Figure 4). Incubation of cisaconitylated melittin at pH 5 for 27 h resulted in only a 30% return in activity, and citraconylated melittin had only a 50% return of activity. Analysis of the kinetics of

Bioconjugate Chem., Vol. 14, No. 1, 2003 55

the cleavage of cis-aconityl modified melittin reveals a rate constant of 0.015 h-1 or a half-life of 47 h, while citraconylated melittin has a half-life of 24 h. The difference between modification by cis-aconitic and citraconic anhydrides may be due to the charge upon modification. As mentioned previously, cis-aconitic modification results in two carboxylate groups, while citraconic modification results in only one carboxylate group. Release of Fluorescein-Labeled Polyethylene Glycol from the Endocytic Compartment by CDMMelittin. To assess the ability of CDM-melittin to function as an endosomolytic agent, its ability to facilitate release of fluorescein-labeled poly(ethylene glycol) (PEG) from endosomes was examined (Figure 5). Similar assays have been used to examine the endosomolytic properties of viruses (33). Cells were incubated with 1 mg/mL fluorescein-PEG ( 400 µg/mL modified melittin for 10 min at 37 °C. After this pulse, cells were washed and chased for an additional 35 min at 37°C in DMEM + 10% bovine serum. In the absence of CDM-melittin, the fluorescein-PEG had a punctuate appearance, indicative of localization in endosomes and/or lysosomes (Figure 5A). In contrast, when CDM-melittin was included, there was diffuse fluorescence throughout the cell, indicating release of fluorescein-PEG from internal endosomes/lysosome (Figure 5B). Addition of cis-aconitic modified melittin resulted in the punctate fluorescence from the endocytosis of the fluorophore (Figure 5C), just as in the absence of melittin. The persistence of a punctuate endosomal signal in the CDM-modified melittin indicates that organelle rupture was not 100% efficient. Similar results were observed when fluoresceinlabeled 10kDa dextran was used as the marker molecule (data not shown). In addition, bafilomycin A, an inhibitor of endosomal acidification, inhibited release of the fluorescein-PEG (data not shown). Visual inspection indicated that 400 µg/mL of CDMmelittin had no apparent cytopathic effect, whereas 10 µg/mL unmodified melittin completely destroyed the cells in less than 10 min. Additionally, propidium iodide staining was used to provide a more sensitive indication of cellular toxicity (34). In the cells exposed to CDMmelittin and after the 10 min wash, ∼1% of the cells showed nuclear staining with propidium iodide, which is similar to control samples (data not shown). Thus, CDM modification of melittin enabled endosomal release while substantially reducing melittin’s cellular toxicity. Delivery of Oligonucleotide. To assay the ability of CDM-modified melittin to aid in the delivery of a biologi-

Figure 5. Confocal fluorescent photomicrographs showing the sub-cellular distribution of endocytosed PEG-fluorescein in HeLa cells without any melittin(A), with CDM-modified melittin(B), or with cis-aconitic modified melittin(C). Settings were identical for all three pictures.

56 Bioconjugate Chem., Vol. 14, No. 1, 2003

Rozema et al.

melittin did not increase the delivery of PMO in the presence of serum. CONCLUSIONS

Figure 6. The effect of 2.5 nmol of maleamate modified melittin on PMO delivery as measured by their induction of luciferase expression in a HeLa cell line stably transformed with the luciferase gene containing an intron with a mutant splice site. The values are normalized to cells treated with PMO alone and represent the means of four separate experiments. The error bars encompass entire range of induction. Data presented for PMO with CDM-melittin (white bar), cis-aconitylated melittin (black bar), and citraconylated melittin (striped bar).

cally active molecule, we chose to study the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs) (35, 36). These agents exert their effects by steric hindrance mechanisms and can be used to block translation or splicing of a target RNA (37-39). PMOs are uncharged nucleotide analogues in which a six-membered morpholine ring is substituted for ribose and an uncharged phosphorodiamidate linkage replaces the phosphodiester linkage (40). Like PEG and dextran, oligonucleotides are internalized by endocytosis (8) and are unable to diffuse across cell membranes (41, 42). The most common method for delivery of anionic oligonucleotides is the use of cationic lipids (43, 44) and polymers (45). However, to use these strategies for the delivery of uncharged oligonucleotides, such as PMO’s, they must first be complexed with a complimentary strand of anionic oligonucleotide to form a charged complex. Two other methods for delivering PMOs, scrapeloading and syringe-loading, both involve physically damaging cells to create transient lesions in the plasma membrane (37) and do not give consistent results. To assay the delivery of PMO by CDM-melittin, we used a commercially available HeLa cell line that carries an integrated luciferase gene with a mutant splice site (39). This mutant splice site results in production of a mRNA coding for a truncated inactive luciferase protein. Blocking this splice site with an oligonucleotide enables expression of the full-length active enzyme. Thus, the luciferase activity in this cell line is directly proportional to the amount of PMO released from the endosomal compartment. Co-incubation of 2.5 µM blocking PMO with 2.5 µM CDM-melittin resulted in 5-12-fold increase in luciferase expression above incubation with PMO alone (Figure 6, white bar). The CDM-melittin facilitated induction was comparable to the induction we obtained using scrape-loading. Neither cis-aconitylated nor citraconylated melittin resulted in any increase in PMO delivery (Figure 6, black and striped bars respectively), supporting our hypothesis that these modifications are too stable to aid in endosome disruption. The presence of 10% serum decreased the amount of delivery observed for CDM-modified melittin and increased the amount of peptide required for optimal delivery. For example, incubation of 2.5 nmol PMO with 5.0 nmol CDM-modified melittin resulted in a 4-5-fold increase in luciferase production. As observed in the absence of serum, citraconylated and cis-aconitylated

The research presented in this report is a demonstration of the concept of endosomolysis by masking of a membrane-active agent (EMMA). This study demonstrates the ability of a new chemical reagent, CDM, to reversibly inhibit the membrane active peptide melittin and enable endosomal release. Melittin is only representative of the many membrane active compounds whose activity can be potentially controlled in order to deliver impermeable compounds. CDM modification could also be used to reversibly inhibit other amine-containing molecules, including membrane-active peptides or polymers. Further studies are in progress to incorporate the CDM modification and CDM-melittin into a variety of synthetic delivery systems for the delivery of oligonucleotides, plasmid DNA and other membrane impermeable drugs. Delivery of the morpholino-based oliogonucleotide by CDM-modified melittin relies on the co-endocytosis of oligonucleotide and melittin. This event is possible in the relatively high concentration of the culture dish, but is impossible in vivo. In order for CDM-based EMMA to facilitate delivery in vivo, the masked membrane-active agent must be associated, with the compound to be delivered. This association may occur by covalent attachment of the membrane-active agent to the compound to be delivered or noncovalent attachment via electrostatic interaction, for example in DNA-polycation complexes. Studies are in progress to attach compounds to be delivered to the masked endosomolytic agent. ACKNOWLEDGMENT

The authors would like to thank Lori Higgs for the synthesis of melittin. We would also like to thank the National Institute of Standards and Technology for financial support through Advanced Technology Program (ATP). LITERATURE CITED (1) Brown, M. D., Schatslein, A. G., and Uchegbu, I. F. (2001) Gene delivery with synthetic (non viral) carriers. Int. J. Pharm. 229, 1-21. (2) Lloyd, J. B. (2000) Lysosome membrane permeability: implications for drug Del.ery. Adv. Drug Del. Rev. 41, 189200. (3) Nishikawa, M., and Huang, L. (2001) Nonviral vectors in the new millenium: delivery Barrier in Gene Transfer. Human Gene Ther. 12, 861-870. (4) Drummond, D. C., Zignani, M., and Leroux, J.-C. (2000) Current status of pH-sensitive liposomes in drug delivery. Prog. Lipid Res. 39, 409-460. (5) Eckert, D. M., and Kim, P. S. (2001) Mechanisms of viral membrane fusion and its inhibition. Annu. Rev. Biochem. 70, 777-810. (6) Nemerow, G. R., and Stewart, P. L. (1999) Role of alpha v integrins in adenovirus cell entry and gene delivery. Microbiol. Mol. Biol. Rev. 63, 725-734. (7) Asokan, A., and Cho, M. J. (2002) Exploitation of intracellular pH gradients in the cellular delivery of macromolecules. J. Pharm. Sci. 91, 903-913. (8) Carrasco, L. (1994) Entry of animal viruses and macromolecules into cells. FEBS Lett. 350, 151-4. (9) Clague, M. J. (1998) Molecular aspects of the endocytic pathway. Biochem. J. 336, 271-282. (10) Gruenberg, J. (2001) The endocytic pathway: A mosaic of domains. Nat. Rev.: Mol. Cell. Biol. 2, 721-730.

Endosomolysis by Masking of a Membrane-Active Agent (11) Mukherjee, S., Ghosh, R. N., and Maxfield, F. R. (1997) Endocytosis. Physiol. Rev. 77, 759-803. (12) Ciftci, K., and Levy, R. J. (2001) Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts. Int. J. Pharm.. 218, 81-92. (13) Kichler, A., Leborgne, C., Coeytaux, E., and Danos, O. (2001) Polyethylenimine-mediated gene Delivery: a mechanistic study. J. Gene Med. 3, 135-144. (14) Klemm, A. R., Young, D., and Lloyd, J. B. (1998) Effects of polyethyleneimine on endocytosis and lysosome stability. Biochem. Pharm. 56, 41-46. (15) Zuber, G., et al. (2001) Towards synthetic viruses. Adv. Drug Del. Rev. 52, 245-53. (16) Budker, V., et al. (1996) pH-sensitive, cationic liposomes: a new synthetic virus-like vector. Nat. Biotech. 14, 760-764. (17) Sitaram, N., and Nagaraj, R. (1999) Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity. Biochim. Biophys. Acta 1462, 29-54. (18) Cheung, C. Y., Murthy, N., Stayton, P. S., and Hoffman, A. S. (2001) A pH-sensitive polymer that enhances cationic lipid-mediated gene transfer. Bioconjugate Chem. 12, 906910. (19) Plank, C., Zauner, W., and Wagner, E. (1998) Application of membrane-active peptides for drug and gene delivery across cellular membranes. Adv. Drug Del. Rev. 34, 21-35. (20) Guo, X., and Szoka, F. C. (2001) Steric Stabilization of Fusogenic Liposomes by a Low-pH Sensitive PEG-Diortho Ester-Lipid Conjugate. Bioconjugate Chem. 12, 291-300. (21) Reddy, J. A., and Low, P. S. (2000) Enhanced folate receptor mediated gene therapy using a novel pH-sensitive lipid formulation. J. Controlled Release 64, 27-37. (22) Blattler, W. A., Kuenzi, B. S., Lambert, J. M., and Senter, P. D. (1985) New Heterobifunctional Protein Cross-Linking Reagent That Forms an Acid-Labile Link. Biochemistry 24, 1517-1524. (23) Naganawa, A., Ichikawa, Y., and Isobe, M. T. (1994) Synthetic Studies on Tautomycin Synthesis of 2,3-Disubstituted Maleic Anhydride Segment. Tetrahedron 50, 8969. (24) Trubetskoy, V. S., et al. (1999) Quantitative assessment of DNA condensation. Anal. Biochem. 267, 309-13. (25) Tang, M. X., and Szoka, F. C. (1997) The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 4, 823-32. (26) Lackey, C. A., et al. (1999) Hemolytic Activity of pHResponsive Polymer-Streptavidin Bioconjugates. Bioconjugate Chem. 10, 401. (27) Wolff, J. A., et al. (1990) Direct gene transfer into mouse muscle in vivo. Science 247, 1465-8. (28) Kirby, A. J., and Lancaster, P. W. (1972) Structure and Efficiency in Intramolecular and Enzymic Catalysis. Catalysis of Amide Hydrolysis by the Carboxy-group of Substituted Maleamic Acids. J. Chem. Soc., Perkin Trans. 2 1206-1214. (29) Nieto, M. A., and Palacian, E. (1983) Effects of temperature and pH on the regeneration of the amino groups of ovalbumin

Bioconjugate Chem., Vol. 14, No. 1, 2003 57 after modification with citraconic and dimethylmaleic anhydrides. Biochim. Biophys Acta 749, 204-210. (30) Kirby, A. J. (1980) Effective Molarities for Intramolecular Reactions. Adv. Phys. Org. Chem. 17, 183-278. (31) Dempsey, C. E. (1990) The actions of melittin on membranes. Biochim. Biophys. Acta 1031, 143-161. (32) Blondelle, S. E., and Houghten, R. A. (1991) Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin. Biochemistry 30, 4671-8. (33) Yoshimura, A. (1985) Adenovirus-induced leakage of coendocytosed macromolecules into the cytosol. Cell Struct. Funct. 10, 391-404. (34) Budker, V., et al. (1997) Protein/amphipathic polyamine complexes enable highly efficient transfection with minimal toxicity. Biotechniques 23, 139, 142-147. (35) Heasman, J., Kofron, M., and Wylie, C. (2000) Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222, 124-34. (36) Nasevicius, A., and Ekker, S. C. (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genetics 26, 216-20. (37) Ghosh, C., and Iversen, P. L. (2000) Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers. Antisense Nucleic Acid Drug Dev. 10, 263-74. (38) Giles, R. V., Spiller, D. G., Clark, R. E., and Tidd, D. M. (1999) Antisense morpholino oligonucleotide analogue induces missplicing of C-myc mRNA. Antisense Nucleic Acid Drug Dev. 9, 213-20. (39) 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, 6235-9. (40) Summerton, J., and Weller, D. (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187-95. (41) Akhtar, S., Basu, S., Wickstrom, E., and Juliano, R. L. (1991) Interactions of antisense DNA oligonucleotide analogues with phospholipid membranes (liposomes). Nucleic Acids Res. 19, 5551-9. (42) Akhtar, S., et al. (2000) The delivery of antisense therapeutics. Adv. Drug Del. Rev. 44, 3-21. (43) Audouy, S., and Hoekstra, D. (2001) Cationic lipid-mediated transfection in vitro and in vivo. Mol. Membr. Biol. 18, 129-43. (44) Hope, M. J., Mui, B., Ansell, S., and Ahkong, Q. F. (1998) Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (Review). Mol. Mem. Biol. 15, 1-14. (45) Robaczewska, M., et al. (2001) Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver. Gene Ther. 8, 874-881.

BC0255945