(Received for publication, January 4, 1996; and in revised form, February 7, 1996)
From the
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.
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) ()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.
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
cells/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 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.
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 50 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 NHCl and monitored OVA
delivery. After treatment with 10 mM NH
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
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
Cl treatment, and the levels of
antigen presentation by the pH-insensitive and -sensitive formulations
were the same in the presence of NH
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 NHCl and compared with the values from untreated
cells. NH
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
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.