Elastase activated liposomal delivery to nucleated cells
`
`Charles C. Pak, Ravi K. Erukulla, Patrick L. Ahl, Andrew S. Jano¡, Paul Meers *
`
`The Liposome Company, 1 Research Way, Princeton, NJ 08540, USA
`
`Received 5 August 1998; received in revised form 9 December 1998; accepted 9 December 1998
`
`Abstract
`
`The specific activation of liposomes for delivery has been explored by enzyme mediated cleavage of a peptide substrate
`covalently conjugated to a fusogenic lipid. We have previously shown an elastase sensitive peptide conjugated to 1,2-dioleoyl-
`sn-glycero-3-phosphatidylethanolamine (DOPE) could be activated by enzymatic cleavage, triggering liposome-liposome
`lipid mixing and fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13^27). Further
`optimization of this system has been aimed at obtaining substrate cleavage at or below physiological elastase levels and to
`demonstrate triggered delivery to living cells. Therefore a new peptide-lipid, MeO-suc-AAPV-DOPE (N-methoxy-succinyl-
`Ala-Ala-Pro-Val-DOPE), has been developed that exhibits greater sensitivity and selectivity for elastase cleavage and
`subsequent conversion to DOPE. This peptide-lipid was used with DODAP (dioleoyl dimethylammonium propane, a pH
`dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposome with an overall
`positive charge if enzymatic cleavage had occurred. Elastase treated liposomes displayed pH dependent enhancement of
`binding, lipid mixing, and delivery of 10 000 MW dextrans, relative to untreated liposomes, when incubated with HL60
`human leukemic cells. Heat denatured elastase did not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating
`enzymatic activity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be
`activated by elastase to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm. These results suggest
`enzyme substrate peptides linked to a fusogenic lipid may be used to elicit specific delivery from liposomes to cells. (cid:223) 1999
`Elsevier Science B.V. All rights reserved.
`
`Keywords: Liposome; Fusion; Elastase; Peptide; Lipid; Cleavage
`
`1. Introduction
`
`A paradox of liposomal delivery vehicles is the
`
`requirement for liposomal membranes that are both
`stable and yet capable of undergoing fusion or desta-
`bilization at the desired site, so as to elicit delivery of
`
`0005-2736 / 99 / $ ^ see front matter (cid:223) 1999 Elsevier Science B.V. All rights reserved.
`PII: S 0 0 0 5 - 2 7 3 6 ( 9 8 ) 0 0 2 5 6 - 9
`
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`112
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`C.C. Pak et al. / Biochimica et Biophysica Acta 1419 (1999) 111^126
`
`the contents of the liposome into the cell. We have
`previously described the use of enzymatic cleavage to
`trigger liposomal fusion and contents delivery [1].
`Although other triggers have been utilized to induce
`liposomal fusion or membrane destabilization [2^7],
`activation by enzyme cleavage has several advantages
`over these methods.
`Elevated enzyme activity is associated with numer-
`ous pathological conditions. Metastatic cancer cells
`display enhanced extracellular activity of several deg-
`radative enzymes, such as matrix metalloproteinases
`and urokinase-type plasminogen activator (for re-
`view see [8]). Elevated enzymatic activity facilitates
`the extravasation of these cells from the circulation
`and increases their invasive potential. In£ammatory
`conditions such as cystic ¢brosis [9^12], rheumatoid
`arthritis [13,14], and emphysema [15,16] are accom-
`panied by an increase in extracellular elastase activity
`due to release of elastase from phagocytic cells. Ele-
`vated elastase activity appears to be due, in part, to
`an imbalance in the elastase/anti-protease ratio
`[9,17,18]. Elastase has also been associated with tu-
`mor progression and development [19^21]. The ubiq-
`uitous yet speci¢c nature of disease-associated enzy-
`matic activity,
`its
`localization near or on the
`membranes of cells involved in tissue remodeling
`[22,23] and its association with several pathologies
`provide numerous opportunities for triggering specif-
`ic liposomal delivery to desired targets using the ac-
`tivity of such enzymes. The triggering event would be
`expected to convert the liposome from a relatively
`inert state to a fusogenic state and may even trigger
`speci¢c binding depending on the design.
`The selectivity of
`liposomal activation can be
`modulated by the choice of an enzyme substrate con-
`jugated to a fusogenic lipid so that enzymatic cleav-
`age releases or unmasks fusogenic lipids. Thus lip-
`osomes may be designed for a selected site of
`activation and hence liposomal delivery could be tar-
`geted. We have chosen to use elastase mediated trig-
`gering of liposomal fusion as a model for the general
`principle of enzymatically activated delivery via lip-
`osomes.
`We have previously described the activation of lip-
`osomal lipid mixing and fusion by enzyme triggering
`[1]. A peptide-lipid consisting of N-Ac-Ala-Ala-, an
`elastase sensitive peptide sequence, was conjugated to
`the headgroup of DOPE (1,2-dioleoyl-sn-glycero-3-
`
`phosphatidylethanolamine), a known fusogenic lipid.
`The fusogenic potential of this peptide-lipid, N-Ac-
`AA-DOPE, was limited until enzymatic cleavage of
`the peptide regenerated DOPE. Liposomes contain-
`ing N-Ac-AA-DOPE could be triggered to lipid mix
`with target liposomes and fuse with RBC ghosts.
`Although N-Ac-AA-DOPE was capable of demon-
`strating the utility of enzyme-triggered fusion, phys-
`iological relevance requires greater sensitivity to en-
`zyme activation, demonstration of delivery to
`nucleated living cells and ultimately the ability to
`deliver in the physiological milieu which may include
`serum proteins. We chose to address the ¢rst two of
`these problems in this report.
`First, it is necessary to demonstrate that the choice
`of an appropriate peptide can be used to optimize
`the cleavage and triggering for the desired target at
`physiological levels of the enzyme. Powers et al. [24]
`had shown that the peptide sequence MeO-suc-Ala-
`Ala-Pro-Val- was highly sensitive to elastase cleav-
`age. Therefore we hypothesized that this sequence
`when conjugated to DOPE would create a trigger-
`able peptide-lipid with greater sensitivity. In this
`study a new peptide-lipid, MeO-suc-AAPV-DOPE,
`was tested for elastase mediated DOPE generation.
`Included in this e¡ort was the introduction of a lipid
`that would reduce the surface charge of the liposome
`compared to the previous version to enhance inter-
`action of the positively charged enzyme, elastase,
`with the substrate. A second goal was to demonstrate
`delivery to nucleated cells, where at least some deliv-
`ery may occur via an endosomal compartment. Lip-
`osomes were designed to take advantage of the low
`pH in that compartment in a manner that would also
`enhance elastase triggering. Here we report a liposo-
`mal system that could be shown for the ¢rst time, to
`be triggered by physiological
`levels of elastase to
`undergo lipid mixing with and aqueous contents de-
`livery to nucleated cells.
`
`2. Materials and methods
`
`2.1. Reagents
`
`Human leukocyte elastase was purchased from
`Calbiochem (San Diego, CA, USA). Lipids were pur-
`chased from Avanti Polar Lipids (Alabaster, AL,
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`113
`
`USA) and were of 99% or greater purity. Calcein
`( s 95% pure),
`tetramethyl
`rhodamine dextran
`(TMR-dextran), and streptavidin were from Molec-
`ular Probes (Eugene, OR, USA). Biotinylated-wheat
`germ agglutinin (WGA) was obtained from Pierce
`(Rockford,
`IL, USA). MeO-suc-Ala-Ala-Pro-Val-
`OH used for covalent
`linkage with DOPE was
`from Bachem Bioscience (King of Prussia, PA,
`USA). RPMI 1640, fetal bovine serum (FBS), and
`HBSS (Hanks’ balanced salt solution) were pur-
`chased from Life Technologies (Gaithersburg, MD,
`USA). Medium 199 was purchased from BioWhit-
`taker (Walkersville, MD, USA). 1,3-Dicyclohexyl-
`carbodiimide
`(DCC),
`4-dimethylaminopyridine
`(DMAP), p-nitrophenol and triethylamine were ob-
`tained from Sigma (St. Louis, MO, USA). TMD-8
`ion exchange resin was purchased from Aldrich (Mil-
`waukee, WI, USA). HPLC-grade solvents, tetrahy-
`drofuran (THF), chloroform and methanol were pur-
`chased from Baxter (McGaw Park, IL, USA). All
`chemicals and solvents were used without further
`puri¢cation.
`
`2.2. Cells
`
`HL60 human leukemia and ECV304 human endo-
`thelial cells were obtained from ATCC (Rockville,
`MD, USA). HL60 cells were passaged as suspension
`cultures in RPMI 1640 supplemented with 10% heat
`inactivated FBS. Adherent ECV304 cells were grown
`in medium 199 supplemented with 10% heat inacti-
`vated FBS. Greater than 98% viability was observed
`during routine tissue culture.
`
`2.3. Synthesis and characterization of N-methoxy-
`succinyl alanine alanine proline valine 1,2-
`dioleoyl-sn-glycero-3-phosphatidylethanolamine
`(MeO-suc-AAPV-DOPE)
`
`2.3.1. MeO-suc-AAPV-p-nitrophenyl ester
`To a solution of MeO-suc-AAPV-H peptide (540
`mg, 1.15 mmol) was added 142 mg (1.38 mmol) of p-
`nitrophenol, 175 mg (1.38 mmol) of DCC and a cat-
`alytic amount (a few crystals) of DMAP in 10 ml of
`dry chloroform. The reaction mixture was stirred
`overnight under nitrogen atmosphere at room tem-
`perature. At this point TLC (thin layer chromatog-
`raphy) analysis showed that the reaction had gone to
`
`completion. The precipitate, dicyclohexylurea, from
`the reaction mixture was ¢ltered using a G-2 funnel
`and the ¢ltrate concentrated under reduced pressure.
`The residual material was used in next step without
`puri¢cation.
`
`2.3.2. MeO-suc-AAPV-1,2-dioleoyl-sn-glycero-
`3-phosphatidylethanolamine
`To a solution of p-nitrophenyl ester of MeO-suc-
`AAPV-OH (600 mg, 1.01 mmol) was added 604 mg
`(0.81 mmol) of 1,2-dioleoyl-sn-glycero-3-phospho-
`ethanolamine and 82 mg (113 ml, 0.81 mmol) of tri-
`ethylamine in 20 ml of chloroform:tetrahydrofuran
`(1:4 v/v). The reaction mixture was stirred under
`nitrogen atmosphere at room temperature overnight.
`TLC analysis showed that the reaction had gone to
`the completion. The reaction mixture was concen-
`trated under reduced pressure and passed through
`activated TMD-8 ion exchange resin in THF:H2O
`(9:1 v/v). The phosphorus positive fractions were
`pooled and concentrated to get a residual product.
`The residual material was puri¢ed with silica gel col-
`umn chromatography (the column was washed with
`5% methanol in chloroform, then eluted with chloro-
`form:methanol:ammonium
`hydroxide
`65:25:4
`v/v/v), giving 915 mg (95% yield on the basis of
`DOPE), which on lyophilization gave a white solid.
`The lipopeptide molecule tested positive with a mo-
`lybdenum reagent and negative with a ninhydrin
`reagent. By TLC the lipopeptide gave a single spot
`and s 99% purity. The lipopeptide was also charac-
`terized by NMR and FAB mass spec analysis. Some
`characteristic 1H-NMR signals (300 MHz, CDCl3)
`are shown here: d 0.87 (t, 3H, J = 7.15 Hz), 1.27
`(40H), 1.56 (4H), 2.0 (8H), 2.23 (t, 4H, J = 7.15
`Hz), 5.17 (1H), 5.32 (4H, J = 3.12 Hz). The 31P-
`NMR spectrum (121.5 MHz, CDCl3) gave a single
`signal. FAB (M(cid:135)) calculated for C62H109N5O15P was
`1195.55, and masses of 1196.8 (MH(cid:135)) and 1234.9
`(MK(cid:135)) were observed.
`
`2.4. Liposome preparation
`
`Large vesicles were prepared by aliquoting desired
`amounts of
`lipid from chloroform stocks
`into
`13U100 mm pyrex tubes and drying under a nitro-
`gen stream. After exposure to high vacuum 4 h
`overnight the lipid ¢lm was hydrated in 10 mM
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`114
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`
`(N-tris[hydroxymethyl]methyl-2-aminoethane-
`TES
`sulfonic acid), 154 mM NaCl, 0.1 mM EDTA (ethyl-
`enediamine tetraacetic acid), pH 7.4. Unless speci¢ed
`otherwise, this bu¡er was used for all experiments.
`After vortexing, samples were freeze-thawed 8U and
`extruded under pressure 10U (Lipex, Vancouver,
`BC, Canada) through 0.1 Wm polycarbonate ¢lters
`(Nucleopore, Pleasanton, CA, USA). Liposomes
`were stored at 4‡C until used.
`Fluorescent dextran containing liposomes were
`prepared by hydrating
`the DODAP/MeO-suc-
`AAPV-DOPE lipid ¢lm with a 50 mg/ml solution
`of 10 000 MW TMR-dextran in TES/NaCl/EDTA
`bu¡er. Liposomes were then vortexed, freeze-thawed,
`and extruded through 0.1 Wm ¢lters as described
`above. To remove unencapsulated dextran the lipo-
`some solution was extensively dialyzed with the TES/
`NaCl/EDTA bu¡er using a Biodialyser (Sialomed,
`Columbia, MD, USA) ¢tted with 50 nm pore size
`¢lters. Calcein loaded liposomes were prepared by
`hydrating the lipid ¢lm in the presence of bu¡er con-
`taining 50 mM calcein. The calcein solution was pH
`and osmolarity adjusted prior to preparation of lip-
`osomes. Calcein loaded liposomes contained 0.75
`mol% N-Rho-PE to monitor liposome binding. After
`preparation of large vesicles as described above, cal-
`cein loaded liposomes were transferred to a 10 000
`MWCO Slide-A-Lyzer (Pierce, Rockford, IL, USA)
`and extensively dialyzed with TES/NaCl/EDTA bu¡-
`er. The encapsulated volume of these liposomes was
`0.8 l/mol of lipid. Sonicated vesicles were prepared
`by drying lipid in the same manner as described
`above but preparations were vortexed then water
`bath sonicated for s 10 min at room temperature.
`Lipid concentration was monitored by phosphate as-
`say [25]. The size of liposomes was veri¢ed by quasi-
`elastic light scattering using a Nicomp submicron
`particle sizer (Particle Sizing Systems, Santa Barbara,
`CA, USA). Freeze-thaw-extrusion vesicles and soni-
`cated vesicles were 70^80 nm and 35^45 nm in diam-
`eter, respectively, as determined by number weighted
`Gaussian analysis.
`
`2.5. Detection of MeO-suc-AAPV-DOPE cleavage
`
`2.5.1. TLC detection of MeO-suc-AAPV-DOPE
`cleavage
`100 nmol of MeO-suc-AAPV-DOPE sonicated
`
`vesicles were incubated with 0, 5, or 10 Wg elastase
`in 0.4 ml TES/NaCl/EDTA bu¡er, pH 7.4, overnight
`at 37‡C. Lipid was extracted by organic phase sepa-
`ration [26], dried under N2 stream, and exposed to
`vacuum for 4 h. Samples were resuspended in chloro-
`form and spotted onto TLC plates. TLC was run
`using chloroform:methanol:ammonium hydroxide
`(65:25:5), air dried,
`sprayed with molybdenate
`blue, and charred on a hot plate.
`
`2.5.2. 31P-NMR analysis
`DODAP/MeO-suc-AAPV-DOPE (1:1 mol ratio)
`freeze-thaw-extrusion vesicles were prepared and
`treated with or without elastase (0^50 Wg protein/
`1000 nmol lipid/4 ml) for 2 h in TES/NaCl/EDTA
`bu¡er, pH 7.4, at 37‡C. Liposomes were transferred
`to 13U64 mm polyallomer centrifuge tubes (Beck-
`man, Palo Alto, CA, USA), and pelleted by ultra-
`centrifugation at 149 000Ug for 1 h at 4‡C with a
`L5-50E ultracentrifuge (Beckman, Palo Alto, CA,
`USA). The liposome pellet (approximately 90% of
`the total) was resuspended in 100 Wl TES/NaCl/
`EDTA bu¡er, to which 400 Wl of 10% deoxycholate,
`100 mM EDTA, 20 mM HEPES, pH 7.4, bu¡er and
`200 Wl of deuterium oxide (Cambridge Isotope Lab-
`oratories, Woburn, MA, USA) were added. After
`transfer to 5 mm NMR tubes samples were moni-
`tored at room temperature in a Bruker AC300 spec-
`trometer operating at 121.5 MHz, with 110 Ws 90‡
`radio frequency pulse for proton decoupling and set
`to 2 s interpulse delay to avoid signal saturation.
`Sweep width was set at 50 kHz. 1 Hz line broadening
`was applied to all spectra. Peaks were identi¢ed by
`comparison with standards run under identical con-
`ditions.
`
`2.6. Binding and lipid mixing of liposomes to HL60
`cells
`
`Lipid mixing was monitored by the N-NBD-PE/N-
`Rho-PE resonance energy transfer assay, as de-
`scribed [27]. Liposomes were prepared with 0.75
`mol% N-NBD-PE and 0.75 mol% N-Rho-PE, which
`results in quenching of the N-NBD-PE £uorescence
`signal. Membrane fusion results in probe di¡usion
`and relief from self-quenching, which is monitored
`as an increase in N-NBD-PE £uorescence. DO-
`DAP/MeO-suc-AAPV-DOPE (1:1 mol:mol)
`lipo-
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`115
`
`somes were incubated in TES/NaCl/EDTA bu¡er (cid:254)
`elastase (5 Wg/100 nmol lipid, 250 WM lipid concen-
`tration) for 2 h at 37‡C, pH 7.4. HL60 cells were
`washed with TES/NaCl/EDTA bu¡er and incubated
`with liposomes (1U106 cells, 10 nmol liposome) in
`200 Wl TES/NaCl/EDTA bu¡er. Samples were either
`at pH 7.4 or adjusted to pH 5 by the addition of
`dilute HCl. All samples were shaken in an Eppendorf
`Thermomixer (Brinkmann Instruments, Inc., West-
`bury, NY, USA), 700 rpm, for 30 min at 37‡C. There
`was no reduction in cell viability following this pro-
`cedure, as detected by trypan blue exclusion (unpub-
`lished data). Cells were then washed with TES/NaCl/
`EDTA bu¡er, pH 7.4, and transferred to Falcon 24
`well plates (Becton Dickinson, Lincoln Park, NJ,
`USA). Fluorescence was monitored in a Cyto£uor
`II multiwell £uorescence plate reader (Perseptive Bio-
`systems, Framingham, MA, USA) with a quartz
`halogen lamp using 450 nm excitation/530 nm emis-
`sion or 560 nm excitation/620 nm emission wave-
`lengths for N-NBD-PE or N-Rho-PE £uorescence,
`respectively. Liposome binding was determined as
`the amount of N-Rho-PE £uorescence associated
`with washed cells relative to total £uorescence of lip-
`osomes added. This percentage was converted to
`number of liposomes bound by multiplying by the
`number of liposomes added (assuming all liposomes
`were 100 nm in diameter and 105 lipid molecules/0.1
`Wm diameter liposome. Therefore 6.02U1010 lipo-
`somes of 0.1 Wm diameter were added per sample).
`The % £uorescence dequenching (FDQ) was calcu-
`lated by the following formula:
`(cid:137)(cid:133)F t=F max cells(cid:134)3(cid:133)F o alone=F max alone (cid:134)(cid:138)=
`(cid:137)13(cid:133)F o alone=F max alone(cid:134)(cid:138)U100
`where Ft = N-NBD-PE £uorescence of liposomes in-
`cubated with cells at a given time, Fo alone = initial
`N-NBD-PE £uorescence of liposomes only, Fmax cells
`and Fmax alone = maximal N-NBD-PE £uorescence of
`liposomes incubated either with cells or alone, re-
`spectively, as determined by addition of 0.5%
`C12E8 detergent. FDQ was assumed to result from
`all-or-none lipid mixing of
`liposomes with cells.
`Therefore % FDQ could be converted to number
`of liposomes mixed by simple multiplication of the
`total. This was done to take into account both the
`
`enhancement of binding and the lipid mixing after
`elastase activation.
`
`2.7. DODAP/MeO-suc-AAPV-DOPE
`liposome-ECV304 binding, lipid mixing, and
`calcein delivery
`
`Liposomes were bound to adherent ECV304 cells
`via a biotin-streptavidin linkage. To this end DO-
`DAP/MeO-suc-AAPV-DOPE (1:1 mol:mol)
`lipo-
`somes were prepared with 0.3 mol% N-biotinyl cap-
`roylamine-PE as well as £uorescent lipid probes or
`with encapsulated calcein. ECV304 cells were washed
`with HBSS bu¡er and then incubated sequentially at
`room temperature with biotin-WGA (20 Wg/ml) and
`streptavidin (40 Wg/ml) prepared in HBSS, 30 min/
`treatment. Cells were washed after each treatment.
`Liposomes were treated with or without elastase as
`described above. Certain aliquots of pretreated DO-
`DAP/MeO-suc-AAPV-DOPE (1:1 mol/mol)
`lipo-
`somes were freeze-thawed after dialysis and prior to
`the addition to cells to release the liposomal con-
`tents. Such freeze-thawed liposomes were exposed
`to a liquid nitrogen-37‡C water bath for 5 cycles.
`The self-quenching of calcein was reduced by ap-
`proximately 85% (maximal FDQ determined by de-
`tergent solubilization) after freeze-thawing, indicat-
`ing the release of encapsulated calcein. In all cases,
`50^100 nmol of liposomes were added to con£uent
`ECV304 cell monolayers (1U105 cells/well of a 24
`well plate) and incubated in HBSS for 30 min at
`room temperature to promote N-biotinyl cap-PE
`binding to streptavidin. Unbound liposomes were re-
`moved by repeated washes. After the ¢nal wash,
`fresh HBSS bu¡er was added to all wells and cells
`were incubated at 37‡C for given times. Fluorescence
`was quantitated as described above.
`
`2.8. Fluorescence microscopy of liposome-cell lipid
`mixing and aqueous contents delivery
`
`DODAP/MeO-suc-AAPV-DOPE (1:1 mol/mol)
`liposomes were incubated for 2 h at 37‡C without
`or with elastase (5 Wg protein/100 nmol lipid). Lipo-
`somes containing the £uorescent lipid probes N-
`NBD-PE and N-Rho-PE were bound to HL60 in
`solution as described above. TMR-dextran loaded
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`
`(40
`liposomes
`DODAP/MeO-suc-AAPV-DOPE
`nmol) were incubated with 1U105 HL60 cells in
`200 Wl TES/NaCl/EDTA bu¡er under pH 5, 37‡C,
`conditions for 30 min to induce binding. Calcein
`loaded DODAP/MeO-suc-AAPV-DOPE liposomes
`were bound to ECV304 cells that had been plated
`on glass coverslips by the biotinylated-WGA-strepta-
`vidin linkage described above. In some experiments
`liposomes were freeze-thawed, as described above,
`prior to the addition to cells. Cells were washed
`with HBSS to remove unbound liposomes and ob-
`served with a Bio-Rad (Hercules, CA, USA) MRC-
`1000
`laser
`scanning
`confocal
`imaging
`system
`equipped with an Olympus BX50 microscope (Olym-
`pus, Lake Success, NY, USA) using an apochromat
`60U oil (N.A. 1.40) objective. Calcein £uorescence
`was excited with the 488 nm line of a krypton/argon
`laser and observed at 522 nm emission. N-Rho-PE
`and TMR-dextran £uorescence were observed with
`the 568 nm line and observed at 605 nm emission.
`Images were frame averaged and false color was ap-
`plied. All images within a ¢gure were obtained under
`identical conditions of confocal iris width, gain, and
`black level. Identical false color look up tables were
`also applied to images within a ¢gure. Average £uo-
`rescence/cell Wm2 of all cells in an image was deter-
`mined with the histogram feature of Bio-Rad Co-
`MOS confocal imaging software.
`
`3. Results
`
`3.1. Cleavage of MeO-suc-AAPV-DOPE by elastase
`
`Cleavage by elastase to yield DOPE is necessary
`for MeO-suc-AAPV-DOPE to act as a trigger for
`liposomal delivery. This was tested by incubating
`MeO-suc-AAPV-DOPE sonicated vesicles with or
`without elastase overnight at 37‡C, after which lipid
`was isolated and analyzed by TLC. Incubation with
`either 5 or 10 Wg elastase/100 nmol lipid resulted in
`the signi¢cant generation of DOPE (Fig. 1), although
`there also appeared to be a slower running phos-
`phate positive component that may represent an in-
`termediate peptide cleavage product. Only minimal
`cleavage of MeO-suc-AAPV-DOPE by proteinase
`K was observed at the same concentrations (unpub-
`lished data), indicating the tetrapeptide sequence is a
`
`Fig. 1. Elastase mediated cleavage of MeO-suc-AAPV-DOPE as
`detected by TLC. 100 nmol of MeO-suc-AAPV-DOPE soni-
`cated vesicles were incubated with 0, 5, or 10 Wg elastase in 0.4
`ml bu¡er. After overnight incubation at 37‡C lipid was ex-
`tracted and spotted onto TLC plates. Plates were run with a
`chloroform:methanol:ammonium hydroxide (65:25:5)
`solvent
`system and visualized by charring. Lane 1: MeO-suc-AAPV-
`DOPE only; lane 2: +5 Wg elastase;
`lane 3: +10 Wg elastase;
`lane 4: 20 Wg DOPE.
`
`more selective target than the previously studied
`N-Ac-AA-dipeptide [1].
`The
`conversion of MeO-suc-AAPV-DOPE to
`DOPE was also quantitated by 31P-NMR. Since sub-
`sequent experiments employed liposomes containing
`both DODAP and MeO-suc-AAPV-DOPE, vesicles
`with a 1:1 (mol:mol) ratio of these two components
`were prepared by the freeze-thaw-extrusion method
`(see Section 2) and incubated at 37‡C for 2 h with
`increasing amounts of elastase (0^5 Wg elastase/100
`nmol lipid). 31P-NMR analysis demonstrated an elas-
`tase concentration dependent cleavage of MeO-suc-
`AAPV-DOPE and appearance of DOPE (Fig. 2, sol-
`id line). A small shoulder that may represent an in-
`complete peptide cleavage product was also observed
`near the original peptide-lipid peak (unpublished
`data). Treatment with 5 Wg elastase/100 nmol lipid
`yielded 20% DOPE. Longer incubation may have led
`to further digestion, though multiple lamellae and/or
`the surface charge of the liposome may limit the
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`117
`
`AAPV-DOPE to DOPE is within concentrations
`that are physiologically or therapeutically relevant,
`with the caveat that the e¡ect of serum proteins
`must still be established.
`
`3.2. Optimum DODAP/MeO-suc-AAPV-DOPE
`composition for binding and lipid mixing with
`HL60 cells
`
`MeO-suc-AAPV-DOPE containing liposomes were
`designed to deliver their contents after binding, en-
`docytic internalization, and fusion with and/or dis-
`ruption of the endosomal membrane. DODAP was
`chosen instead of the previously used DOTAP [1]
`because only 20% of the DODAP population is pos-
`itively charged at pH 7.4 [29]. This allows a more
`
`Fig. 3. Optimum DODAP/MeO-suc-AAPV-DOPE liposome
`composition for elastase activation of binding/lipid mixing with
`HL60 cells. DODAP/MeO-suc-AAPV-DOPE liposomes pre-
`pared at 1:3, 1:1, or 3:1 (mol/mol) ratios and labeled with 0.75
`mol% N-NBD-PE and 0.75 mol% N-Rho-PE were incubated
`with or without elastase (5 Wg elastase/100 nmol lipid) for 2 h
`at 37‡C. 10 nmol of liposomes were then mixed with 1U106
`HL60 cells and incubated for 30 min at 37‡C, pH 5. After
`washing twice with 5U volume of TES/NaCl/EDTA bu¡er, pH
`7.4, (A) binding and (B) lipid mixing was determined by moni-
`toring N-Rho-PE and N-NBD-PE £uorescence, respectively.
`
`Fig. 2. Quantitation of elastase mediated cleavage of MeO-suc-
`AAPV-DOPE to DOPE by 31P-NMR. DODAP/MeO-suc-
`AAPV-DOPE (1:1 mol/mol) freeze-thaw-extrusion vesicles were
`incubated with 0, 0.5, 1, 2, 5 Wg elastase/100 nmol lipid. Sam-
`ples were incubated for 2 h at 37‡C, after which liposomes
`were pelleted by ultracentrifugation. Liposomes were solubilized
`and monitored by 31P-NMR. Solid line: % of total MeO-suc-
`AAPV-DOPE converted to DOPE; dotted line: % of expected
`MeO-suc-AAPV-DOPE on outer monolayers
`converted to
`DOPE assuming unilamellar vesicles, i.e. result multiplied by 2.
`
`ultimate amount digested (see Section 4). The max-
`imum exposed peptide-lipid for intact
`liposomes
`would occur with unilamellar vesicles. Assuming
`only the outer lea£et peptide-lipid is available for
`digestion, the minimal percentage conversion of ex-
`posed MeO-suc-AAPV-DOPE to DOPE in the outer
`lea£et lipid was 40% in this situation (Fig. 2, dotted
`line). If the average number of lamellae were greater
`than one, the percentage conversion was even higher.
`In fact, the ratio of encapsulated volume to total
`lipid would appear to indicate an average of approx-
`imately 2.5 lamellae per vesicle for this preparation
`(see Section 2) which would indicate that 100% of
`available peptide-lipid had been cleaved under these
`conditions. The concentration of elastase required to
`produce this amount of cleavage (12.5 Wg elastase/ml)
`is less than the amount of elastase activity found in
`the epithelial lining £uid from patients with cystic
`¢brosis [28]. The contents from human neutrophil
`granules were also able to cleave MeO-suc-AAPV-
`DOPE and generate DOPE (unpublished data).
`The number of neutrophils required to observe this
`e¡ect was less than that observed in epithelial lining
`£uid from cystic ¢brosis patients [28], indicating the
`amount of elastase required to cleave MeO-suc-
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`118
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`C.C. Pak et al. / Biochimica et Biophysica Acta 1419 (1999) 111^126
`
`complete peptide hydrolysis by elastase (unpublished
`data, see Section 4) so that we can model the more
`general case of cleavage on the liposome surface that
`is not a¡ected by the high charge of the enzyme. For
`other enzymatic activators it may be possible to uti-
`lize a permanently positively charged lipid that
`would allow the liposome to become positively
`charged at physiological pH after peptide cleavage,
`so that extracellular binding would be triggered. By
`contrast,
`the tertiary amine of DODAP would
`be fully protonated at pH 5, suggesting liposomes
`containing DODAP would undergo stronger inter-
`action with negatively charged cell membranes within
`the low pH environment of the endocytic compart-
`ment.
`In order to determine the optimum combination of
`DODAP and MeO-suc-AAPV-DOPE for triggerable
`binding and lipid mixing with cells the two lipids
`were formulated into liposomes at di¡erent ratios.
`DODAP/MeO-suc-AAPV-DOPE liposomes
`pre-
`pared at 1:3, 1:1, and 3:1 mol ratios were pretreated
`with or without elastase and incubated with HL60
`cells under low pH conditions to promote DODAP
`mediated binding to cells. Only DODAP/MeO-suc-
`AAPV-DOPE liposomes at a 1:1 mol ratio exhibited
`an elastase dependent increase in binding and lipid
`mixing with HL60 cells (Fig. 3), possibly as a result
`of increased positive charge after enzymatic cleavage.
`Apparently the amount of DODAP in 1:3 liposomes
`was insu⁄cient to mediate binding to cells, even at
`pH 5 after elastase treatment. By contrast, DODAP/
`MeO-suc-AAPV-DOPE (3:1 mol ratio) liposomes
`were able to bind to cells with or without elastase
`treatment, re£ecting the greater amount of DODAP
`in these liposomes. These liposomes were also able to
`lipid mix with cells even without elastase activation.
`The DODAP/MeO-suc-AAPV-DOPE 1:1 mol ratio
`liposomal formulation was chosen for all further
`studies in order to develop a delivery system that
`can be triggered by enzymatic cleavage.
`
`3.3. Elastase activated binding and lipid mixing of
`DODAP/MeO-suc-AAPV-DOPE liposomes with
`HL60 cells
`
`Since DODAP was included in liposomes with
`MeO-suc-AAPV-DOPE to enhance binding with
`cells under
`low pH conditions, we determined
`
`Fig. 4. pH dependence of DODAP/MeO-suc-AAPV-DOPE
`liposome binding/lipid mixing with HL60 cells. Fluorescent lipid
`probe labeled DODAP/MeO-suc-AAPV-DOPE liposomes were
`incubated with or without elastase (5 Wg elastase/100 nmol lip-
`id) for 2 h at 37‡C. 10 nmol of liposomes were mixed with
`1U106 HL60 cells in 200 Wl TES/NaCl/EDTA bu¡er. Samples
`were incubated for 30 min at 37‡C, at
`the given pH and
`washed. Binding (A) and lipid mixing (B) were determined by
`monitoring N-Rho-PE and N-NBD-PE £uorescence, respec-
`tively.
`
`whether the pH dependence of DODAP mediated
`binding is within physiological
`levels. DODAP/
`MeO-suc-AAPV-DOPE (1:1) liposomes were pre-
`treated with elastase and incubated with HL60 cells
`at di¡erent pH. Enhanced binding and lipid mixing
`of elastase pretreated liposomes with HL60 cells were
`observed when incubated at pH 4.6 or pH 5.1 (Fig.
`4). Incubation at pH 5.8^7.4 did not yield any sig-
`ni¢cant association of liposomes with cells. These
`results suggest that these liposomes are sensitive to
`elastase mediated activation of binding and lipid
`mixing when DODAP is maximally positively
`charged. The pH required to achieve this state is
`present under normal physiological conditions within
`the late endosome [30].
`To con¢rm that the enzymatic activity of elastase
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`C.C. Pak et al. / Biochimica et Biophysica Acta 1419 (1999) 111^126
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`119
`
`microscopy of liposomes with HL60 cells con¢rmed
`that elastase pretreatment was required for enhanced
`binding and lipid mixing. Bright £uorescent labeled
`cells indicate the N-Rho-PE £uorescent probe from
`the bound elastase activated liposomes had mixed
`into the cell plasma membrane (Fig. 6). In contrast,
`untreated liposomes displayed signi¢cantly less bind-
`ing and lipid mixing (Fig. 6). Quantitation of £uo-
`rescence images revealed nearly 12 times as much
`£uorescence/cell area in HL60 cells that had been
`incubated with DODAP/MeO-suc-AAPV-DOPE lip-
`osomes treated with elastase, as compared to those
`cells incubated with untreated liposomes, although
`the distribution of this enhanced delivery may not
`be uniform across all the cells. Almost no £uores-
`cence was associated with the cells at pH 7.4 (unpub-
`lished data).
`
`3.4. Aqueous contents delivery from
`DODAP/MeO-suc-AAPV-DOPE liposomes to
`HL60 cells
`
`To determine if the enhanced lipid mixing between
`elastase activated DODAP/MeO-suc-AAPV-DOPE
`liposomes and HL60 cells is truly indicative of fu-
`sion, it is necessary to monitor the delivery of an
`aqueous probe from the liposome to the cell cyto-
`plasm. Therefore DODAP/MeO-suc-AAPV-DOPE
`liposomes were loaded with tetramethyl rhodamine
`labeled 10 000 MW dextran (TMR-dextran), treated
`with or without elastase, and incubated with HL60
`cells under pH 5 conditions.
`Only DODAP/MeO-suc-AAPV-DOPE liposomes
`that had been pretreated with elastase were capable
`of fusing with HL60 cells, as demonstrated by TMR-
`dextran labeling of the cytoplasm of these cells (Fig.
`7). HL60 cells incubated with liposomes that had not
`been treated with elastase contained little or no cy-
`toplasmic £uorescent dextran,
`indicating elastase
`cleavage was required to trigger the fusion of DO-
`DAP/MeO-suc-AAPV-DOPE liposomes with HL60
`cells. TMR-dextran delivery to