©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Delivery of Macromolecules into Cytosol Using Liposomes Containing Hemolysin from Listeria monocytogenes(*)

(Received for publication, January 4, 1996; and in revised form, February 7, 1996)

Kyung-Dall Lee (§) Yu-Kyoung Oh Daniel A. Portnoy (1) Joel A. Swanson (§)

From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo.


INTRODUCTION

Liposomes are potentially universal carriers of various molecules, particularly in vivo(1, 2, 3) . Thus far, however, liposome-mediated delivery into the cytosolic space has been inefficient. More often, membrane-impermeant and high molecular weight molecules delivered inside liposomes are degraded inside macrophage lysosomes, without ever crossing endocytic membranes into cytoplasm(4, 5) . Currently, the best available formulation is the ``pH-sensitive liposome,'' which maintains stable phospholipid bilayers at neutral pH but favors non-bilayer structure at the acidic pH of endosomes (for a review, see (6) ). Although delivery from pH-sensitive liposomes into the cytosolic space was better than that achieved by conventional, pH-insensitive liposomes, there remains a significant need for improved mechanisms that can mediate delivery of the liposomal contents into the cytosol after release within endocytic compartments(6, 7, 8) .

The efficiency of cytoplasmic delivery could be greatly enhanced by a mechanism that breaches the endosomal membrane once the liposomal contents are delivered into endosomal lumen. For this, we have adopted a strategy used by a facultative intracellular pathogen, Listeria monocytogenes(9) . After being internalized into phagosomes, L. monocytogenes permeabilizes phagosomal membranes to enter the cytosolic space of host cells(9) . Listeriolysin O (LLO) (^1)is primarily responsible for this step(10, 11, 12) , although other factors may augment its action(13, 14, 15) . Purified LLO is hemolytic and shows increased activity at low pH(16, 17) . Here, we report co-encapsulation of LLO and other molecules to be delivered inside liposomes and demonstrate that LLO can confer upon liposomes, both pH-insensitive and -sensitive, the ability to deliver efficiently their contents into macrophage cytosol.


EXPERIMENTAL PROCEDURES

Murine bone marrow-derived macrophages were prepared as described in Racoosin and Swanson(18) . Cells were cultured in Dulbecco's minimal essential medium containing 20% fetal calf serum and 30% L-cell-conditioned medium. On the sixth or seventh day of culture, cells were replated and cultured on coverslips or appropriate dishes in 10% fetal calf serum containing Dulbecco's minimal essential medium for use the following day.

LLO was purified from L. monocytogenes strain M1545 according to a method modified from Goldfine et al. and Geoffroy et al.(14, 16) . Briefly, bacterial culture supernatant was concentrated by tangential flow ultrafiltration (30-kDa molecular cutoff filters, Minitan system, Millipore, Bedford, MA), followed by fractionation using successive anion and cation exchange columns (Q-Sepharose; MonoS, Pharmacia Biotech Inc.). The protein eluant peak from a cation exchange column (NaCl gradient) containing hemolytic activity was pure by Coomassiestained SDS-polyacrylamide gel electrophoresis. The protein migrated at 58 kDa.

Liposomes were made by freeze-thaw followed either by extrusion through 0.2 µm size polycarbonate filters (Nuclepore Costar, Cambridge, MA) four times or by sonication(19, 20) . LLO was encapsulated inside liposomes as soluble protein in 30 mM Tris buffer, 100 mM NaCl, at pH 8.5, under non-reducing conditions. Unencapsulated molecules and LLO were removed by gel filtration (Sepharose CL, Pharmacia, Piscataway, NJ). The liposomes used for morphological studies contained 35 mM 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) and 50 mMp-xylene-bispyridinium bromide (DPX) (Molecular Probes, Eugene, OR). For antigen presentation, ovalbumin (OVA) (Sigma) was encapsulated inside liposomes at 20 mg/ml; and LLO was at 200 µg/ml (100 to 1 molar ratio of OVA to LLO). The amount of encapsulated OVA was monitored using OVA labeled with fluorescein isothiocyanate. Under the highest OVA concentration we tested in the antigen presentation assay, 100 µg/ml, the lipid concentration was approximately 500 µM and the LLO, 1 µg/ml. LLO-containing liposomes were stable over 2 weeks as indicated by a calcein release assay(4) .

The antigen presentation assay was according to a modified method of Harding(21) . In brief, after incubation with liposomes for 1 h at 37 °C, macrophages were washed and returned to normal culture medium for 2 h. Macrophages were then fixed in 1% paraformaldehyde, washed again, and then incubated with CD8 T cells (CD8 OVA T1.3 cell line, 10^6cells/ml) for 20-24 h. Interleukin-2 content in the supernatant was measured by enzyme-linked immunosorbent assay (Genzyme, Cambridge, MA). All the experiments with drug treatments were done in the cells pretreated with drugs for 15 min before incubation with liposomes.

Phagocytic index of macrophages toward IgG-opsonized sheep erythrocytes (SE) was measured according to the method of Wright and Silverstein (22) . Cells treated with liposomes were incubated with SE opsonized with anti-SE IgG at a 10:1 ratio (SE/cell) for 5 min at 37 °C. Then, unbound SE were washed and the bound but non-internalized SE were lysed by 3 times alternating 2-s washes with buffer and water. Cells were subsequently fixed, and the number of macrophages containing SE and the number of SE per positive cell were scored.


RESULTS AND DISCUSSION

We tested two lipid compositions: A, pH-insensitive liposomes, which consisted of phosphatidylcholine (PC) and cholesterylhemisuccinate (CHEMS); and B, pH-sensitive liposomes, made of phosphatidylethanolamine (PE) and CHEMS (Table 1). Both have the same negative charge density at neutral pH and exhibit similar rates of uptake by macrophages(7) . Formulations denoted C and D (Table 1) were the same as A and B, respectively, except that they contained encapsulated LLO. Hereafter, we refer to the LLO-containing liposomes as ``primed'' liposomes.



The cytoplasmic delivery by pH-sensitive, primed liposomes was first visualized directly by fluorescence microscopy. The membrane-impermeant, pH-dependent, fluorescent dye HPTS was encapsulated inside liposomes along with DPX, which at high concentrations quenches the fluorescence of HPTS(6, 19) . Fig. 1(a-d) shows HPTS fluorescence excited at 405 and 440 nm, inside macrophages that internalized liposomes. At low pH, HPTS fluorescence is greater with 405 nm excitation, whereas at neutral pH, fluorescence at 440 nm excitation is greater than at 405 nm excitation(5, 6, 19) . Therefore, a strong fluorescence at 405 nm excitation indicates HPTS in acidic compartments (endosomes); and a strong signal at 440 nm indicates HPTS in neutral compartments (cytosol). Cells incubated with pH-sensitive, primed liposomes showed HPTS fluorescence in the cytoplasm, often brightly labeling the nucleus (Fig. 1, c and d), whereas cells incubated with pH-sensitive, unprimed liposomes showed predominantly endosomal, low pH HPTS signal (Fig. 1, a and b). Within 30 min after a 15-min incubation with liposomes, 54 (±16)% of cells incubated with pH-sensitive, primed liposomes showed bright cytoplasmic HPTS fluorescence, in contrast with 14 (±10)% positive using pH-sensitive, unprimed liposomes (n = 4; each approx50 cells). Furthermore, in the majority of positive cells, the cytoplasmic fluorescence in the cells treated with primed liposomes was stronger than that with unprimed liposomes.


Figure 1: a-d, delivery of liposome-encapsulated fluorescent dye by LLO-containing liposomes. Fluorescence of cells incubated with HPTS/DPX containing liposomes: a and b, pH-sensitive, unprimed B formulation; c and d, pH-sensitive, primed (i.e. LLO-containing) D formulation. a and c were with excitation at 405 nm (20 nm bandpass), and b and d with 440 nm (20 nm bandpass); all at emission 520 nm. Liposomes containing HPTS/DPX were incubated with bone marrow-derived macrophages for 15 min at 37 °C. Cells were washed and chased for an additional 15 min at 37 °C before viewing. e and f, a time lapse sequence of HPTS delivery by pH-sensitive, primed liposomes. The uptake and fate of HPTS/DPX-containing liposomes (formulation D) was monitored using an image analysis system (Universal Imaging, West Chester, PA) equipped with alternating excitation filters (e excited by 405 nm; f excited by 440 nm). After 8 min of incubation with liposomes, cells were washed with buffer, and the fluorescence at two excitation wavelengths was collected every minute. Frames represent 1-min intervals, beginning 5 min after the wash.



The sequence from a time lapse movie indicated the mechanism of delivery from the pH-sensitive primed liposomes (Fig. 1, e and f). Release of HPTS from liposomes into endocytic compartments, and subsequently into cytosol, was monitored microscopically, using two alternating fluorescence excitation filters. During the initial period of incubation with liposomes, most cells showed low HPTS fluorescence, because HPTS fluorescence was quenched by DPX inside intact liposomes. Upon destabilization of liposomes and release of liposomal contents inside the endocytic compartment, HPTS fluorescence was dequenched, resulting in an increased signal at 405 nm excitation (Fig. 1e, frame 2). After a lag time of 1 min, HPTS diffused rapidly from the acidic compartment into the cytosol, producing a diffuse signal throughout the cytoplasm (Fig. 1f). The cytoplasmic signal was stronger at 440 nm excitation than at 405 nm excitation, demonstrating that the dye had entered the pH-neutral compartment (i.e. cytosol). This last step was only observed with primed liposomes indicating an important role for LLO in the efficient cytoplasmic delivery.

For the LLO-containing liposomes to be effective as a therapeutic delivery vehicle, cells must remain viable after delivery. We measured the viability of cells after liposome uptake in two ways. First, we examined the permeability of plasma membranes to propidium iodide and trypan blue. After incubation with liposomes for 1.5 h, cells were returned to normal culture medium, and dye exclusion was monitored 1.5 and 24 h later. All liposome formulations showed less than 6% cells positive with dyes, and these values were similar to cells treated with buffer only. As a second test, we monitored the phagocytic index of macrophages toward IgG-opsonized SE(22) . None of the liposome formulations affected either the percentage of cells that phagocytosed SE or the average number of SE phagocytosed per macrophage (Table 2).



To quantify cytosolic delivery of macromolecules by the pH-sensitive, primed liposomes relative to other liposomes, we used an assay for MHC class I-restricted antigen presentation(21) . Exogenous proteins internalized by macrophages are normally processed by proteolysis in endocytic compartments and presented at the cell surface in an MHC class II-restricted response; MHC class I-restricted presentation is induced predominantly when proteins enter cytosol(8, 23, 24) . We encapsulated OVA inside liposomes, added them to macrophages, and then measured the extent of OVA antigen presentation via cytosolic pathway, using a CD8 T cell line that recognizes OVA peptide-MHC I complex on macrophage cell surfaces(21) . OVA delivered into the cytosol is degraded by proteasome-mediated proteolysis in the cytoplasm, the processed peptide is translocated into the endoplasmic reticulum, where it associates with a MHC class I molecule, and this peptide-MHC complex is then transported to the cell surface via the Golgi apparatus.

Primed liposomes, both pH-insensitive and -sensitive, delivered OVA into the cytosolic pathway of antigen presentation far more efficiently than corresponding unprimed formulations. Antigen presentation was most efficient using pH-sensitive, primed liposomes (Fig. 2a, D formulation). Although pH-sensitive liposomes have been previously reported to induce MHC class I-restricted antigen presentation in comparison with pH-insensitive liposomes(8) , neither formulation delivered OVA into cytosol efficiently enough to induce measurable antigen presentation in our assay. This is likely due to the different sensitivities of the assay methods. Antigen presentation was not detectable when macrophages were allowed to internalize free OVA (100 µg/ml) by pinocytosis, with or without free LLO (1 µg/ml) in the medium (data not shown). In time course experiments (Fig. 2b), the OVA presentation via pH-sensitive, primed liposomes increased after a lag period of 30 min, reached a maximum at 4 h, and then decreased to almost zero after 24 h.


Figure 2: a, OVA-specific MHC class I-restricted antigen presentation by macrophages. Different amounts of OVA encapsulated inside liposomes (formulations A-D as listed in Table 1) were incubated with macrophages, and the extent of OVA-specific antigen presentation was documented by measuring interleukin-2 (IL-2) production from CD8 OVA T1.3 cells incubated with macrophages according to the method as described under ``Experimental Procedures.'' The data are averages of two independent triplicate experiments. b, kinetics of antigen presentation after OVA delivery via pH-sensitive primed liposomes. Experiments were the same as in a, at 33 µg/ml OVA concentration. After incubation with liposomes, cells were kept in medium for different lengths of time before being fixed and tested for antigen presentation.



To examine the pH dependence of delivery, we elevated the pH of endocytic compartments by treating cells with NH(4)Cl and monitored OVA delivery. After treatment with 10 mM NH(4)Cl(25) , OVA antigen presentation by pH-insensitive, primed liposomes was substantially abrogated (Fig. 3a). This most likely reflected the pH dependence of LLO activity(16, 17) ; NH(4)Cl treatment had no effect on the extent of MHC class I-mediated antigen presentation when OVA was directly introduced into the cytoplasm by scrape loading (26) (Fig. 3a). The antigen presentation by the pH-sensitive primed liposomes was more dramatically reduced (99%) by NH(4)Cl treatment, and the levels of antigen presentation by the pH-insensitive and -sensitive formulations were the same in the presence of NH(4)Cl. This indicated to us that one of the principal differences between the two formulations might be the location and timing of the release of liposomal contents along the endocytic pathway. Accordingly, OVA and LLO inside pH-sensitive liposomes would be released in acidic prelysosomal compartments whereas release from the pH-insensitive formulation would occur later, perhaps in lysosomes, consistent with published data on the pH-sensitive liposomes(6, 7) .


Figure 3: a, the effect of elevated endocytic pH. The extent of antigen presentation by macrophages using primed liposomes, pH-insensitive (C) and pH-sensitive (D), was monitored in macrophages treated with 10 mM NH(4)Cl and compared with the values from untreated cells. NH(4)Cl was present during exposure to liposomes and for 2 h afterward, and the concentration of liposomal OVA incubated with cells was 33 µg/ml. The extent of antigen presentation was also monitored in the cells scrape loaded with 3 mg/ml OVA, with and without NH(4)Cl treatment. Scrape loading was performed according to the method described in (26) (n = 3, ±S.D.). IL-2, interleukin-2. b, the effect of proteasome inhibitor. The extent of antigen presentation using formulation (D) was measured when cells were treated with different concentrations of proteasome inhibitor MG132 (a gift from Dr. A. Goldberg, Harvard Medical School). MG132 was solubilized in dimethyl sulfoxide (DMSO) and incubated with cells during the incubation with liposomes and chase period. The extent of antigen presentation with different final concentrations of dimethyl sulfoxide is plotted against control (no treatment) as 100% (solid bars). When MG132 was added at different concentrations under the shown dimethyl sulfoxide concentration, antigen presentation was significantly reduced (hatched bars) (n = 3, ±S.D.). c, the effect of BFA. Different duration of BFA treatment during 1-h liposome incubation and 2-h chase period (pulse-chase scheme diagrammed at the bottom) showed a reversible inhibition of antigen presentation by BFA. The antigen presentation in treated cells was expressed as percent of the value in untreated cells. All BFA treatments included a 15-min pretreatment. Final concentration was 1 µg/ml BFA in 0.01% dimethyl sulfoxide.



To ensure that the observed antigen presentation occurred via the conventional MHC class I pathway, we monitored the effects of inhibitors of two critical steps in the route for antigen presentation: MG132, an inhibitor of proteasomes(27) , and brefeldin A (BFA), an inhibitor of endoplasmic reticulum to Golgi traffic(28) . Antigen presentation of OVA introduced into cytosol by primed liposomes was inhibited by MG132 in a dose-dependent manner (Fig. 3b). Moreover, it was also inhibited by BFA, and this inhibition by BFA was reversible (Fig. 3c). Together, the inhibition by MG132 and BFA indicated that the liposomal OVA was first delivered into the cytoplasm and then followed the conventional MHC class I antigen presentation pathway. The recovery of antigen presentation after BFA block was an additional indication of cell viability after exposure to LLO-containing liposomes.

It is difficult to quantify the percent of internalized macromolecules that reach the cytosolic space. Nonetheless, we have demonstrated that pH-sensitive primed liposomes represent a dramatic improvement over earlier formulations. Based on the data in Fig. 2, we estimate that the antigen presentation efficiency is at least 30-fold higher than that of unprimed pH-sensitive liposomes. It is comparable to antigen presentation mediated by other particulate carriers such as bacteria or beads (29, 30) and perhaps by osmotic lysis of pinosomes(23) . However, a liposome delivery system has better potential than these other delivery systems for in vivo and therapeutic applications.

This is the first report that utilizes a bacterial hemolytic protein to design a liposome formulation that delivers its contents into cytosol by adopting the strategy used by L. monocytogenes to enter into cytoplasm. The toxicity of LLO is minimized by encapsulation inside liposomes. Furthermore, the natural properties of LLO implicate its minimal toxicity compared with other related pore-forming hemolysins(31) . These data further indicate that LLO is sufficient to mediate lysis of endocytic compartments. However, further molecular characterization of the hemolytic function of LLO is needed to improve upon the delivery system we have described here.

One obvious application of these liposomes, implicated by the antigen presentation assay in this report, is as vaccine vectors for cell-mediated immunity. Additionally, these formulations should allow cytoplasmic delivery of any macromolecule that can be encapsulated inside liposomes. Such molecules could include drugs, peptide, or protein regulators of molecular events in the cytoplasm, oligonucleotides, and higher molecular weight nucleic acids. The significance may be greater considering this method can be readily transferred to in vivo models.


FOOTNOTES

*
This work was supported by grants from the National Institutes of Health and the Harvard Medical School Funds for Discovery. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence may be addressed: Dept. of Cell Biology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1642; Fax: 617-432-0407; kdlee{at}warren.med.harvard.edu.

(^1)
The abbreviations used are: LLO, listeriolysin O; OVA, ovalbumin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CHEMS, cholesterylhemisuccinate; HPTS, 8-hydroxypyrene-1,3,6-trisulfonate; DPX, p-xylene-bispyridinium bromide; SE, sheep erythrocytes; BFA, brefeldin A; MHC, major histocompatibility complex.


ACKNOWLEDGEMENTS

We thank Melissa Johnson and Dr. Steven Baer for technical support in image analysis and Drs. Howard Goldfine, Andreas Gallusser, and Tomas Kirchhausen and Eun Ji Shin for help in purification of LLO. We also thank Dr. Alfred Goldberg for the generous gift of MG132 (courtesy of ProScript Inc.) and Dr. C. Harding for the OVA-specific CD8 T cell line.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.