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[Cancer Research 61, 3978-3985, May 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Binding of Novel Peptide Inhibitors of Type IV Collagenases to Phospholipid Membranes and Use in Liposome Targeting to Tumor Cells in Vitro1

Oula Penate Medina, Tim Söderlund, Liisa J. Laakkonen, Esa K. J. Tuominen, Erkki Koivunen and Paavo K. J. Kinnunen2

Helsinki Biophysics and Biomembrane Group, Department of Medical Chemistry, Institute of Biomedicine [O. P. M., T. S., L. J. L., E. K. J. T., P. K. J. K.], and Department of Biosciences, Division of Biochemistry [E. K.], University of Helsinki, Helsinki, FIN-00014, Finland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently described a novel cyclic peptide inhibitor CTTHWGFTLC (CTT) for matrix metalloproteinases (MMP)-2 and MMP-9, also called type IV collagenases or gelatinases (E. Koivunen et al., Nat. Biotechnol., 17: 768–774, 1999). As indicated by its amino acid composition, CTT is hydrophobic, and its partitioning into phospholipid films could be verified by the monolayer technique. Augmented fluorescence emission anisotropy (from 0.064 to 0.349) and reduced collisional quenching by I- of the Trp residue in CTT was evident in the presence of unilamellar phosphatidylcholine/phosphatidylethanolamine liposomes, revealing the association of CTT with the lipid bilayers. Gelatinases are potential targets of therapeutic intervention in cancer, and inhibitors of these enzymes can prevent tumor progression in animal models. CTT enhanced 3- to 4-fold the cellular uptake of liposome-encapsulated water-soluble fluorescent marker, rhodamine B by gelatinase-expressing cells. Gelatinase targeting seems to be essential, as modified peptides that were less potent gelatinase inhibitors were also less efficient in promoting the cellular uptake of liposomes. Augmented killing (~4-fold) of U937 leukemia and HT1080 sarcoma cells was obtained by the CTT-enhanced delivery of Adriamycin-containing liposomes, compared with control liposomes administered without the peptide. These results suggest a novel type of utility for small gelatinase inhibitors in targeted cancer therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MMPs3 represent a family of enzymes capable of degrading the basement membrane and extracellular matrix, thus contributing to tissue remodeling and cell migration (1 , 2) . MMPs can be divided into subgroups, one of which is constituted by the type IV collagenases or gelatinases, MMP-2 and MMP-9 (3) . Expression of gelatinases in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages, is tightly regulated (4) . Similarly to other MMPs, gelatinases are secreted in an inactive form (pro-MMP) and proteolytic cleavage is needed for their activation (5) .

Elevated or unregulated expression of gelatinases and other MMPs can contribute to the pathogenesis of several diseases, including tumor angiogenesis and metastasis (6) , rheumatoid arthritis (7) , multiple sclerosis (8) , and periodontitis (9) . Compounds inactivating gelatinases may thus provide potential therapeutic means for cancer and inflammatory disorders (10 , 11) . Although a number of MMP inhibitors have been described, specific inhibitors of gelatinases have not been available (11) . Recently, we screened random phage peptide libraries with the aim of developing a selective inhibitor against this MMP subgroup. The most active peptide derived, abbreviated CTT, was found to selectively inhibit the activity of MMP-2 and MMP-9 of the MMP family members studied (1) . CTT also inhibited endothelial and tumor cell migration in vitro, as well as tumor progression in vivo, in mouse models, which indicated the importance of gelatinases in tumor invasion.

Experiments in mice bearing tumor xenografts showed that CTT-displaying phages were accumulated in the tumor vasculature after their i.v. injection into the recipient mice. Targeting of the phage to tumors was inhibited by the coadministration of CTT peptide (1) . These results suggest that CTT, besides being a potent antitumor agent itself by blocking cancer cell migration and angiogenesis, may also be used for the targeting of chemotherapeutics to tumors.

In chemotherapy, only a fraction of the therapeutic drug reaches the cancer cells, whereas the rest of the drug may damage normal tissues. Adverse effects can be reduced by the administration of cancer drugs encapsulated in liposomes (12) . Improved liposome compositions have been described so as to enhance their stability and also to prolong their lifetime in the circulation (13) . Both in vitro and in vivo studies on the development of liposomes targeted to cancer cells have been reported (14, 15, 16) . Enhanced selectivity can be obtained by derivatizing the liposomes with specific antibodies that recognize plasma membrane antigens of target cells, thus augmenting the uptake of liposomes by cells (17, 18, 19) . Because both MMP-2 (20 , 21) and MMP-9 (22, 23, 24) are bound by specific cell-surface receptors, these enzymes represent potential receptors for liposome targeting to invasive cells, such as tumor cells and angiogenic endothelial cells.

Inhibitors, complexed with their target proteinases, can be rapidly taken up into cells by endocytosis (25 , 26) . We studied here the efficiency of the gelatinase-targeting CTT peptide to promote the uptake of liposomes by cultured gelatinase-expressing cells. Our data demonstrate that CTT added to liposomes enhances significantly their uptake by such tumor cells in culture, thus providing the possibility for selective liposomal delivery of chemotherapeutic agents.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
eggPC, POPE, Adriamycin (doxorubicin), rhodamine B, and 0.01 M PBS with 2.7 mM KCl and 0.137 M NaCl (pH 7.4) at 25°C (PBS) were purchased from Sigma Chemical Co. (St. Louis, MO), and NBD-PE and POPC from Avanti (Birmingham, AL). The other fluorescent lipid, DPPRho, and the fluorescent probe phenanthridinium, or EthD-1, were from Molecular Probes (Leiden, Netherlands). MMP-2 was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). DMEM and RPMI 1640 with Glutamax-1 were from Life Technologies, Inc. (Paisley, Scotland). Phospholipid stock solutions were made in chloroform. The purity of lipids was checked by TLC on silicic acid-coated plates (Merck, Darmstadt, Germany) using chloroform/methanol/water (65:25:4, v/v/v) as a solvent. Examination of the plates after iodine staining or, when appropriate, on fluorescence illumination revealed no impurities. Concentrations of the nonfluorescent phospholipids were determined gravimetrically using a high-precision electrobalance (Cahn Instruments, Inc., Cerritos, CA), and those of the fluorescent phospholipid analogues were determined spectrophotometrically using the molar extinction coefficients {epsilon} = 93,000 at 540 nm for DPPRho and {epsilon} = 21,000 at 463 nm for NBD-PE, with methanol as a solvent. Antihuman MMP-2 was kindly provided by Dr. Timo Sorsa (Department of Medical Chemistry and Periodontology, University of Helsinki, Helsinki, Finland). Antihuman MMP-9 (M-17, sc-6841) was from Santa Cruz Biotechnology (Santa Cruz, CA). Marimastat was obtained from British Biotech (Oxford, United Kingdom). TIMP-2 and the fluorogenic substrate for gelatinases, MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg, were from Calbiochem (La Jolla, CA).

Sequence Homology Search.
CTT homologues were searched with BLASTP 1.4.11 MP, using strategy (identity matrix, word size one, and expectation value as 1000) recommended for small peptides by National Center of Bioinformation (27, 28, 29, 30) . The other parameters were kept at their default values. Our query sequence was CXXHWGFTXC. Nine analogues were found, among them a part of the EGF-7 domain in human laminin ß-1 chain precursor CLPGHWGFPSC (denoted here as CLP). Homologues to CLP (CLPGHWGFPSC) were also searched for and were compared with the SWISS-PROT SIM program.

Synthetic Peptides.
Peptides were synthesized on an Applied Biosystems 433 A (Foster City, CA) automatic synthesizer using Fmoc-chemistry. Disulfide bridges were formed in 5% acetic acid (pH 6.0) containing 20% DMSO by incubation overnight at room temperature with continuous stirring (31) . After 1:2 dilution with 0.1% trifluoro acetic acid, peptides were loaded onto a preparative reversed-phase high-performance liquid chromatography column and eluted by an acetonitrile gradient. The identity of the peptides was verified by mass spectrometry. The peptides used in this study, with their amino acid sequences and respective abbreviations are compiled in Table 1Citation .


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  		Table 1 Amino acid sequences of CTT, CLP, STT, and CWL with the conserved residues in boldface

 
Assay for Gelatinases.
Inhibition of MMP-9 and MMP-2 by the different synthetic peptides was measured using casein zymography (32) . MMP-9 was purified as described previously (33) . Subsequently, MMP-2 (2.5 µg) or MMP-9 (2.5 µg) was run on a 10% SDS-PAGE containing 2 mg/ml casein. The gel was first washed in Triton X-100 containing buffer to remove SDS, and it was cut into five slices that were immersed into peptide (CTT, CLP, CWL, or STT)-containing solutions (85 µM). After incubation for 48 h at 37°C, the gels were stained with Coomassie Blue and scanned, and the digested areas were quantitated using image analysis (Global Lab Image 3.2; Data Translation Inc. and Acuity Imaging Inc., Marlboro, MA).

The activity of MMP-9 was measured also using the fluorogenic peptide substrate MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg (34) . More specifically, 50 ng of MMP-9 were preincubated in PBS for 30 min at room temperature in the absence or presence of CTT or its liposome complex. Subsequently, the above substrate was added to a final concentration of 0.1 mM, and the incubation was continued at 37°C for 15 min, after which the fluorescence intensities were measured with excitation at 340 nm and emission at 390 nm in a microtiter plate reader.

Interaction of Peptides with Lipid Monolayers.
Lipid monolayers residing on an air-water interface provide a convenient means to assess lipophilicity of peptides by monitoring the increase in surface pressure caused by insertion into the film (35) . Penetration of the indicated peptides into eggPC films was measured using magnetically stirred circular wells (subphase volume, 400 µl). Surface pressure ({pi}) was monitored with a Wilhelmy wire attached to a microbalance (µ; TroughS, Kibron Inc., Helsinki, Finland) and connected to a Pentium personal computer. Lipid was spread on the air-buffer [PBS (pH 7)] interface in chloroform (~1 mg/ml) to different initial surface pressures ({pi}0), and the resulting monolayer was allowed to equilibrate for 15 min before the addition of the peptides (4 µl, 10 mg/ml in H2O, and CWL in DMSO) into the subphase. The increment in {pi} from the initial surface pressures ({pi}0) after the addition of peptide was complete in ~20 min, and the difference between {pi}0 and the value observed after binding of the peptide into the film was taken as {Delta}{pi}. All of the measurements were performed at ambient temperature (~24°C). The data are represented as {Delta}{pi} versus {pi}0 (35) .

Preparation of Liposomes.
Lipid stock solutions were mixed in chloroform to obtain the desired compositions. The solvent was removed under a gentle stream of nitrogen, and the lipid residue was subsequently maintained under reduced pressure for at least 2 h. Multilamellar liposomes were formed by hydrating the dry lipids at room temperature with 1 ml of PBS containing rhodamine B (10 µM) or Adriamycin (1.8 µM) to encapsulate these compounds into liposomes, so as to yield a lipid concentration of 1 mM. Multilamellar liposomes were freeze-thawed five times to enhance encapsulation (36) . LUVs were obtained by extruding (37) multilamellar dispersions 19 times through a 100-nm pore-size polycarbonate membrane (Nucleapore, Pleasanton, CA) with a LiposoFast Pneumatic gas pressure operated small-volume homogenizer (Avestin, Ottawa, Canada). The pressure used for extrusion of vesicles through the filters was 25 psi (~170 kPa). When indicated, the peptides (2 mg/ml in PBS, except CWL in DMSO) were mixed with the LUVs to yield final lipid and peptide concentrations of 1 mM and 0.5 mg/ml, respectively.

Fluorescence Spectroscopy.
The environments of the tryptophan residues of CTT, STT, CLP, and CWL in liposomes were studied by fluorescence spectroscopy. The center of Trp fluorescence peak is at ~350 nm when in water, whereas in a hydrophobic environment, the emission is centered near 330 nm. Accordingly, changes in the microenvironment of Trp can be monitored by measuring I350:I330, the ratio of the emission at 350 nm to that at 330 nm (38) . Tryptophan fluorescence was recorded with a Perkin-Elmer LS 50 B spectrofluorometer equipped with a magnetically stirred and thermostated cuvette compartment. All of the measurements were done at 37°C in 5 mM HEPES, 0.1 mM EDTA (pH 7.4) buffer. Excitation and emission bandpasses were 10 nm and 10 nm, respectively. Excitation wavelength was 295 nm, and emission spectra were recorded in the range 300–400 nm. Trp emission spectra of CTT, STT, CLP, and CWL were recorded both in absence of and presence of POPC/POPE (80/20, mol/mol) LUVs, yielding final peptide and lipid concentrations of 10 µM and 100 µM, respectively.

Fluorescence polarization of the Trp residue of CTT as a function of increasing concentration of liposomes was measured using rotating polarizers in the excitation and emission beams, with a bandpass of 10 nm. The results are expressed as emission anisotropy (38) calculated as follows:

In this equation I{perp} and III represent the emission intensity perpendicular and parallel, respectively, to the excitation polarizer.

The extent of exposure of Trp to the aqueous phase was assessed using I- as a collisional quencher(38) .The extent of quenching was calculated by usinge quation:

where F0 and F are Trp fluorescence intensities at 345 nm in the absence and presence of the quencher, Q; {tau}0 is Trp fluorescence lifetime in the absence of the quencher, k, the Stern-Volmer constant (obtained from the slope of the linear fit of the data). Fluorescence of Trp in CTT (5 µM) and its quenching were measured in the absence and presence of POPC/POPE (80:20, mol/mol) LUVs (final lipid concentration of 0.5 mM). The results were corrected for scattering caused by liposomes in PBS.

Assay for Lipid Mixing.
The ability of the CTT, CLP, STT, and CWL peptides to induce liposome fusion was assessed by measuring lipid mixing, as described earlier (39) . In brief, NBD-PE (X = 0.01) and DPPRho (X = 0.01) were incorporated into liposomes (POPC/POPE, 78/20, mol/mol). Because of the spectral overlap of NBD-PE (donor) emission and DPPRho (acceptor), absorption resonance energy transfer between these dyes is highly efficient. As liposomes containing NBD-PE and DPPRho are mixed with nonlabeled liposomes, fusion is observed as an increase in the emission intensity of NBD-PE caused by the dilution of probes. Excitation and emission wavelengths for NBD-PE at 430 nm and 530 nm were used. All of the fluorescence measurements were done at 37°C.

Assays for Liposome Uptake by Cells.
To study the effects of the different peptides on the cellular uptake of liposomes, DPPRho was incorporated into liposomes as a tracer, to yield a composition POPC/POPE/DPPRho (80:19:1, mol/mol). The indicated peptides were mixed with LUVs and subsequently added to cells cultured in microtiter well plates. After an incubation for 5 min at 38°C liposomes not bound to the cells were removed by rinsing the three times with cold PBS. The relative amount of DPPRho associated with the cells was determined using Tecan Spectrafluor Plus microplate reader (Tecan, Hombrechtigon, Switzerland) with excitation at 535 nm and emission at 595 nm.

In another series of experiments the water soluble fluorescent marker rhodamine B was encapsulated into liposomes and its uptake by cells was measured. Samples for rhodamine B uptake were prepared by mixing 60 µl of POPC/POPE (80/20, mol/mol, 1 mM) liposomes containing 10 µM rhodamine B to 300 µl of DMEM supplemented with 10% fetal bovine serum, Glutamax I, penicillin 100 units/ml, and streptomycin 0.1 mg/ml. Peptides were then added to obtain a final concentration of 8.5 µM. Ten µl (105 cells) of medium containing the indicated cells were combined with the LUVs. After an incubation for 15 min at 37°C or at 4°C, liposomes not bound to the cells were removed from 96-well plates by rinsing three times with cold PBS. The measurement at 4°C served as a control for nonendocytotic liposome uptake by cells (40) . The relative amount of rhodamine B associated with the cells was determined using microplate reader with excitation at 535 nm and emission at 595 nm.

Treatment of cells by TPA has been reported to increase both the expression and secretion of MMP-9 (21) . Accordingly, in some experiments, we preincubated cells with TPA (50 nM) for 15–75 min. Results are presented as RFI = I/I0, where I0 is the emission intensity for the control (2 µM rhodamine B in LUVs without the peptides) and I is the rhodamine emission for the peptide-liposome complexes taken up by the cells. Effects of the indicated antibodies ({alpha}MMP-9 and {alpha}MMP-2) and gelatinase inhibiting compounds (TIMP-2, Marimastat, and EDTA), on the uptake of rhodamine B containing liposomes (POPC/POPE, 80:20 molar ratio) by HT1080 cells was performed as above. M-17 inhibits the binding of MMP-9 to the substrate, and this inhibition is expected to be concentration dependent. The effect of rabbit polyclonal antibody against human MMP-2 has not been studied (34) . Final concentrations of the indicated peptide, Adriamycin, antibodies, and liposomes were 85 µM, 0.2 mM, 20 µg/ml, and 0.4 µM, respectively.

Cell Cultures and Fluorescence Microscopy.
U937 (ECACC 85011440), HT1080 (ECACC 85111505), and CHO (ECACC 85050302) cells were cultured in RPMI medium or DMEM supplemented with 10% fetal bovine serum, Glutamax I, penicillin (100 units/ml), and streptomycin (0.1 mg/ml). Uptake of rhodamine B containing liposomes by the cultured cells was verified by fluorescence microscopy. An inverted fluorescence microscope (Zeiss IM 35) equipped with Nikon extra-long working distance objectives (x20) and (x40) were used. Samples were prepared as described for the rhodamine uptake assay, with slight modifications. Accordingly, instead of being washed, U937, HT1080, and CHO cells, with the rhodamine-containing liposomes and in cell culture medium, were transferred into Nunclon 48-plate wells. Excitation and emission wavelengths were selected by suitable bandpass filters (Melles-Griot, Zevenaar, the Netherlands) transmitting in the range of 535 nm and >600 nm, respectively. Fluorescence images were viewed with a Peltier-cooled digital camera (C4742–95, Hamamatsu, Japan) connected to a computer and operated by software (Hipic 5.0) provided by the instrument manufacturer.

Viability Assay.
Peptide-liposome complexes with the encapsulated Adriamycin were made as described above, yielding final concentrations of peptide, lipid, and Adriamycin of 85 µM, 0.2 mM, and 0.4 µM, respectively, and were subsequently added to a 10-µl (105 cells) sample of U937 cells. For the detection of dead cells, EthD-1 was used. This fluorophore is membrane impermeable and emits fluorescence only when bound to DNA (41) . Accordingly, EthD-1 readily enters dead cells and binds to their DNA, its fluorescence intensity correlating with the amount of dead cells. EthD-1 emission was measured using a microplate reader with excitation at 495 nm and emission at 635 nm. Student’s t test was used to assess statistical significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gelatinase-inhibiting Peptides.
Three peptides were selected for this study (Table 1)Citation . CTT is a recently described cyclic collagenase inhibitor, which has been shown to target tumors (1) . STT is the corresponding linear sequence of CTT in which the terminal cysteines were replaced by serines. The third peptide was the CTT homologue CLP, found by sequence homology search from SWISS-PROT and EMBL databases by Blast 1.4.11 program. Because our main interest was the conserved part of CTT, our query sequence was CXXHWGFTXC (1) . Computer search revealed nine homologues, among them the sequence of CLP, which is a part of EGF-7 domain in human laminin ß-1 chain (LMB1). Three of the other eight homologues were laminins from other species, one was from immunoglobulin heavy chain, and four were thiazide-sensitive sodium-chloride cotransporters from human and other species. Because this latter group had less apparent similarity to CTT, we focused instead on CLP, the CTT-resembling sequence of the laminin EGF-7 domain. Notably, all of the three peptides (CTT,CLP, and STT) inhibited the activity of MMP-2 as assessed by casein zymography (Fig. 1)Citation . The extent of inhibition was [sim[70% at 85 µM CTT. Also, CLP was a potent inhibitor of MMP-2, causing an 50% inhibition at 85 µM peptide. The linear CTT analogue STT (85 µM) reduced the activity of MMP-2 by ~30%. Similar results were also obtained with MMP-9.



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  		Fig. 1. A, inhibition of MMP-2 by CTT, CLP, STT, and CWL peptides each at 85 µM and assessed by casein zymography. Results are shown as the percentage of area digested, for which uninhibited MMP-2 is taken as 100%. Error bars, SDs for three separate experiments. B, inhibition of MMP-9 by free CTT ({circ}) and bound to liposomes (•), measured using the fluorogenic substrate as described in "Materials and Methods." Total concentration of phospholipid was 200 µM POPC/POPE (80/20 mol/mol). Data points, mean values from triplicate experiments; bars, SD.

 
Free CTT and CTT, complexed with liposomes, inhibited MMP-9 also when studied by the gelatinase assay using the fluorogenic peptide as a substrate (Fig. 1B)Citation , in keeping with the results obtained by casein zymography. The IC50 values for CTT and CTT liposome were ~8 µM in this assay. These results additionally demonstrate that the MMP-9 interacting epitope of CTT remains available for interaction with the enzyme when bound to the lipid membrane. Complete inhibition of MMP-9 (5 nM) in this assay was obtained by 100 µM EDTA, which chelates the Zn2+ required for catalysis (data not shown).

Interaction with Phospholipid Monolayers.
The amino acid composition of CTT readily suggests that this peptide is hydrophobic. Accordingly, it was of interest to study whether CTT binds to lipids. For comparison, we also investigated CLP and STT. For this purpose, we used phospholipid monolayers residing on an air-buffer interface, a model biomembrane that has been widely used to study lipid-protein interactions (42 , 43) and the effects of proteins on the lateral organization of lipid monolayers (43 , 44) . Penetration of the peptide into the lipid monolayer after injection underneath the lipid film increases the surface pressure {pi} (45) , whereas peptides that do not bind to lipids cause no changes (35) . Initial surface pressure ({pi}0) of eggPC monolayers was varied between 10 and 40 mN/m, and increments in surface pressure ({Delta}{pi}) attributable to the addition of peptides (final concentration, 200 µM) into the subphase were measured (Fig. 2)Citation . All three of the peptides readily penetrated into the monolayer films, and the slopes of {Delta}{pi} versus {pi}0 were qualitatively similar. At initial monolayer packing pressures exceeding 38, 31, and 33 mN/m for CTT, STT, and CLP, respectively, the membrane penetration of these peptides was abolished.



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  		Fig. 2. Penetration of CTT (•), CLP ({blacksquare}), and STT ({blacktriangleup}) into an eggPC monolayer, evident as an increase in surface pressure ({Delta}{pi}) after the addition of the indicated peptide into the aqueous subphase. Data are shown as a function of the initial surface pressure ({pi}0).

 
Interactions with Liposomes.
The above experiments using eggPC lipid monolayers revealed that CTT, CLP, and STT binded to membranes. This was confirmed by measuring Trp fluorescence emission anisotropy for CTT in the presence of increasing concentrations of liposomes (Fig. 3A)Citation . Accordingly, in the absence of liposomes, the value for r was 0.065, reflecting rapid Brownian rotational diffusion of the peptide in solution. However, increasing concentrations of liposomes caused progressively increasing r, up to 0.349 measured at 230 µM phospholipid. Approximately one-half maximal effect was evident at CTT:phospholipid molar ratio close to 5:90.



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  		Fig. 3. A, anisotropy (r) for the Trp residue of CTT as a function of POPC/POPE (80/20 mol/mol) concentration. Data points, the average from five measurements; error bars, ±SD. To improve signal:noise ratio, the signal was averaged for 15 s. Concentration of CTT was 5 µM in PBS, and temperature was 37°C. B, Trp emission intensity for free CTT peptide ({circ}) and its liposome complex (•) was measured both with and without I- as a water-soluble collisional quencher. Data points, two separate measurements.

 
Intrinsic tryptophan fluorescence allows us to estimate possible changes in the microenvironment of this fluorophore on the association of the peptide with liposomes (38) . Value for I350:I330 measured in PBS for the Trp residue of CTT is 1.4, whereas in the presence of LUVs (0.5 mM total phospholipid), this ratio decreased to 1.00. Trp, thus, resides in a more hydrophobic environment in the presence of liposomes, in keeping with the partitioning of CTT into lipid membranes. In addition, the quenching of Trp by I- was reduced in the presence of liposomes and reveals that the Trp residue in CTT is only partially exposed to the water-soluble collisional quencher I- when the peptide is bound lipids. The Stern-Volmer constants were 0.0087 M-1 and 0.0034 M-1 in the absence and presence of liposomes, respectively.

Lipid-binding peptides and proteins may also induce fusion of lipid vesicles (46) . To explore this possibility, labeled and nonlabeled LUVs were mixed in the absence and presence of peptides, and lipid mixing was measured as described previously (39) . However, neither CTT, STT, nor CLP caused measurable changes in the fluorescence emission intensity of NBD-PE within 15 min, revealing lack of lipid mixing and, thus also, vesicle hemifusion and fusion (data not shown).

Effects on Cellular Uptake of Liposomes.
The above data show that CTT binds to phospholipids and that CTT does not cause liposome fusion. Accordingly, it was of interest to investigate the possibility that CTT could be used in liposome targeting. We first approached this by including a fluorescent lipid marker DPPRho, into liposomes as described in "Materials and Methods." Subsequently, CTT was added to these liposomes, after which they were added to U937 leukemia cells, serving here as a model of gelatinase-expressing cells (47) . After 5 min, the cells were washed, and the fluorescence in cells was measured by microplate reader. CTT promoted the association of the liposomes with U937 cells, and ~3.7-fold more of the fluorescent marker DPPRho was bound to the cells when CTT was present (data not shown).

We then proceeded to study the ability of CTT to enhance the uptake by U937, CHO, NRK52E, and HT1080 cells of the water-soluble fluorescent marker, rhodamine B, encapsulated into liposomes. The same liposomes, but without CTT, were used as a control. Five min after the liposomes with CTT were added to cells, the soluble rhodamine label could be detected in cells by fluorescence microscopy. Expression of MMP-2 or MMP-9 is essential for liposome uptake. Accordingly, CHO cells, which do not express gelatinases according to our zymographic assay, showed no uptake of CTT liposomes under conditions in which an enhanced liposome uptake was evident for both HT1080 and U937 cells (Fig. 4)Citation . Although there was variation in the fluorescence intensities of individual cells, we did not observe cells lacking liposome uptake in the latter two cell lines.



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  		Fig. 4. Fluorescence microscopy images of rhodamine B intake by U937, CHO, and HT1080 cells incubated with POPC/POPE (80:20, molar ratio) liposomes with the encapsulated fluorescent marker and with the targeting peptide, CTT, as indicated. A and B, U937 cells, which have been incubated with liposomes with and without CTT, respectively. C and D, the same experiment for CHO cells (exposure time prolonged 10-fold); E and F, the same experiment for HT1080 cells.

 
Uptake of liposome-encapsulated rhodamine was quantitated by fluorescence plate reader. The uptake of liposome-capsulated rhodamine into U937 cells was enhanced 3.6-fold by CTT, compared with the uptake of liposomes lacking the peptide (Fig. 5A)Citation . Only a minor effect was observed for STT and CLP, and the signal was too weak to be statistically significant in comparison with liposomes lacking the peptides. The scrambled cyclic peptide CWL was also inefficient in promoting the liposome uptake. A similar effect of CTT on liposome uptake was evident for human HT1080 fibrosarcoma and NRK52E rat kidney epithelial-like cells (data not shown). CTT did not promote the uptake of free rhodamine B. Enhanced liposome uptake by CTT was evident only at 37°C but not at 4°C, thus indicating active receptor-mediated endocytosis to be involved (Fig. 5B)Citation . TPA has been observed to enhance both the expression and secretion of MMPs (21) . The effect of TPA on the uptake of CTT liposomes is biphasic, and, after a short exposure time (15 min), liposome intake is increased, whereas after a prolonged incubation (75 min), the uptake of liposomes is inhibited. This could be explained by gelatinase at low concentrations remaining bound mainly to the cell surface, whereas at high concentrations, the excess enzyme is released into the extracellular space (21 , 34) .



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  		Fig. 5. A, effects of the indicated peptides on the uptake of rhodamine B containing liposomes (POPC/POPE, 80:20 molar ratio) by U937 cells. Total lipid, rhodamine B, and peptide concentrations were 200 µM, 2 µM, and 100 µM, respectively. The data are normalized by comparing rhodamine fluorescence of the cells incubated with liposomes with the added peptide (I) to cells incubated with liposomes without the peptides (I0 = control). Error bars, SD (n = 3). B, CTT was added to liposomes encapsulating the soluble fluorescent marker rhodamine B. Uptake of this fluorophore by HT1080 cells was determined after a 30-min incubation with liposomes at 37°C or 4°C. Data points, means ± SD from triplicate wells. C, CTT was added to liposomes containing the fluorescent lipid marker DPPRho. After a 30-min incubation of CTT-liposomes with U937 cells, fluorescence bound to cells was determined. The data are normalized by comparing fluorescein fluorescence of the cells incubated with liposomes with the added peptide (I) with cells incubated with liposomes without CTT (I0 = control).

 
Gelatinases as Targets of CTT Liposome Complexes.
We further investigated whether cell surface-bound gelatinases were the receptors for the CTT-containing liposomes. Cells were washed to remove soluble forms of gelatinases and then were preincubated for 30 min with MMP inhibitors or specific antibodies before the addition of liposomes. These experiments showed that the internalization of CTT-containing liposomes by HT1080 cells could be particularly efficiently prevented by antibodies against MMP-9. In particular two such MMP-9 antibodies, when used together, completely blocked the internalization of the liposome-encapsulated fluorescent dye (Fig. 6)Citation . Inhibition by an antibody to MMP-2 was only partial. An antibody against cytosolic part of integrin ß1 had no effect. These results indicate a specific blockade of the liposome uptake by the anti-MMP-9 and anti-MMP-2 antibodies.



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  		Fig. 6. Effects of the indicated antibodies on the uptake of rhodamine B containing liposomes (POPC/POPE, 80:20 molar ratio) by HT1080 cells. Results are shown as the percentage of rhodamine fluorescence of the cells incubated with liposomes with the added CTT peptide without antibodies in the medium taken as 100%. Antibodies used are anti-MMP-2, anti-MMP-9, and anticytocolic domain of integrin ß3, which was used as a control antibody. Final concentrations of the indicated peptide, Adriamycin, antibodies, and liposomes (expressed as total phospholipid) were 85 µM, 0.2 mM, 20 µg/ml, and 0.4 µM, respectively. Error bars, SDs for four separate experiments.

 
Studies with a panel of proteinase inhibitors revealed that MMP inhibitors, but not serine proteinase inhibitors, prevented the liposome uptake. More specifically, MMP inhibitors TIMP-2 and Marimastat and the cation-chelator EDTA all had a similar effect, causing a [SIM]50% inhibition of liposome transfer into cells cultured in 10% FCS (Fig. 7)Citation , whereas serum trypsin inhibitors or aprotinin were practically without effect.



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  		Fig. 7. Effects of the indicated gelatinase inhibitors on the uptake of rhodamine B-containing liposomes (POPC/POPE, 80:20 molar ratio) by HT1080 cells. Results are shown as the percentage of rhodamine fluorescence of the cells incubated with liposomes with the added CTT peptide without the antibodies in the medium taken as 100%. Inhibitor used here is TIMP-2 (10 µg/ml). Marimastat (50 µM) is a MMP family-inhibiting synthetic drug, EDTA is a Zn2+ gelator (500 µM), and aprotinin (1 µg/ml) is a serine protease inhibitor. Final concentrations of the indicated peptide, rhodamine, and liposomes (expressed as total phospholipid) were 85 µM, 0.2 mM, and 0.4 µM, respectively; error bars, SD for four separate experiments.

 
Adriamycin, a widely used anticancer drug, has been encapsulated into liposomes with high efficiency (48) .The efficiency of CTT to promote liposome uptake and to target this therapeutic agent to tumor cells was studied in U937 culture. CTT was added to the liposome solution to a 200-µM concentration, corresponding to a CTT:phospholipid molar ratio of ~1:2. This solution was then added to U937 cells to yield final CTT and Adriamycin concentrations of 85 µM and 0.4 µM, respectively. Up to the concentration levels used in this study the synthetic peptides and liposomes as such were not toxic to the cells (data not shown). Liposomes without added CTT were used as a control, and cell-killing was assessed by the EthD-1 assay. After 24 h, a 4.1-fold increase in killing of the cells (P < 0.001) was observed in comparison to liposomes without CTT (Fig. 8)Citation . Also gelatinase-inhibiting peptides STT and CLP, but not the scrambled CWL peptide, enhanced cell killing by liposome-encapsulated Adriamycin, except to a lesser degree than with CTT. Accordingly, STT and CLP increased the number of dead cells 1.7- and 1.3-fold (P < 0.01), respectively.



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  		Fig. 8. Comparison of the effects of CTT, CLP, STT, and CWL on U937 cell killing induced by Adriamycin-containing liposomes and assessed by EthD-1 fluorescence. Cells were incubated with Adriamycin (200 ng/ml) encapsulated in liposomes (200 µM total phospholipid; POPC/POPE, 80:20 molar ratio) and the indicated peptides. Final concentrations of the indicated peptide, Adriamycin, and liposomes (expressed as phospholipid) were 85 µM, 0.2 mM, and 0.4 µM. Results are expressed as RFI/RFI0, where RFI0 is the value for cells incubated with Adriamycin-containing liposomes without the peptides. Concentration of the peptides was 85 µM. Bars, SDs (n = 3).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to their physiological expression and binding to specific cell-surface receptors in normal cells, gelatinases are also abundantly present in invasive and metastatic tumor cells (49 , 22) . Gelatinases also play a role in the angiogenesis and affect the formation of the new blood vessels nurturing the tumor (24) . These enzymes thus provide potential targets for more selective delivery of liposomes and drugs to tumors. In accordance with these findings, CTT, which is a selective cyclic decapeptide inhibitor of gelatinases found by screening phage display libraries, prevents growth of human tumor xenografts in mice and targets the tumor vasculature after an i.v. injection (1) . Liposomes encapsulated with a cancer drug could provide an effective approach to selectively kill the tumor vasculature providing that the liposomes can be targeted to the site of action of gelatinase, e.g., by using CTT as a homing peptide.

The present results show that CTT binds to phospholipids. This could be demonstrated by surface pressure studies on lipid monolayers. The critical pressure in monolayers, 38 mN/m, needed to prevent the penetration of CTT into films, is high and reflects the highly amphiphilic nature of this peptide. The 5.4-fold increment in Trp emission anisotropy for CTT measured in the presence of liposomes (230 µM) confirmed this peptide to bind to the bilayer. The spectral features of Trp emission remained unaltered when CTT was added to liposomes, which indicated a lack of changes in the microenvironment of this fluorophore. This indicates that the Trp in CTT remains exposed to the aqueous phase also when bound to liposomes. Accordingly, the HWGF sequence of CTT, which is important for the gelatinase inhibitory activity (1) , may remain available for interactions with the enzyme active site also when CTT is associated with liposomes. CTT did not cause liposome fusion, which thus enables its use in liposome targeting.

The above properties of CTT prompted us to study the possibility that this peptide could be used in the targeting of liposomes to gelatinase-expressing cells. This was found to be feasible, and a ~3- to 4-fold enhanced uptake of liposomes U937 and HT1080 cells was observed by using fluorescent tracers. On the other hand, CTT was without effect when CHO cells were used. Because the latter do express gelatinases to a lesser extent, it appears that the presence of these enzymes is a prerequisite for the liposome uptake-promoting effect of CTT. This was indicated by the inhibition of the uptake observed in experiments with anti-MMP-2 and anti-MMP-9 antibodies and the MMP inhibitors TIMP-2, Marimastat, and EDTA. The complete inhibition exerted by anti-MMP-9 antibodies in HT1080 cells suggests that MMP-9 is the major target of CTT, at least in this cell line. Thus, the effect of the peptide on invasivity and migration of HT1080 cells (1) is also likely to be caused by the targeting of MMP-9. Other studies have similarly suggested that HT1080 cells strongly depend on MMP-9 for migration (50) . In other cell types, MMP-2 could be the more important target of CTT. Augmented liposome internalization attributable to CTT was seen already within 15 min after the addition of liposomes and was evident only at 37°C but not at 4°C. Accordingly, MMP-2 and MMP-9 seem to provide the ligand-binding sites for CTT-containing liposomes that are required for the fast endocytotic process, which could thus provide an efficient route for directing drugs to rapidly proliferating cancer cells.

Comparison of peptides (i.e., CTT, CLP, and STT) that differ in their ability to inhibit gelatinase activities reveals a correlation between the gelatinase inhibitory effect and the efficiency to mediate liposome binding to cells. More specifically, the efficiencies of the peptides in liposome targeting decreased in the order of CTT>CLP>STT, which is the same as their gelatinase-inhibiting efficiency. This correlation suggests that cell surface-associated gelatinases are the receptors for the CTT peptide-liposomes. Because sequences similar to CTT are present in laminin, it is also possible that cell-surface receptors for laminin may play a role in peptide-liposome binding and internalization. However, CLP that was derived from laminin was only moderately efficient in liposome targeting. Furthermore, the CTT sequence does not resemble known cell-binding sequences of laminin (51) . On the other hand, the fact that CLP is a moderately efficient gelatinase inhibitor suggests the possibility for the regulation of gelatinases by laminin EGF-like fragments. Gelatinases by itself degrade laminins (52 , 53) , and could then be inhibited by the fragments generated as an end point of cell migration. Our results thus suggest that MMP-2 and MMP-9 may participate in a rapid endocytotic process, representing a novel target for therapeutic intervention.

CTT and liposomes as such were nontoxic to cultured cells. An important finding reported here is that there was a 4-fold increase in the efficiency of killing cultured human cells by Adriamycin that contained liposomes when CTT was added to the liposomes. Interestingly, unlike the situation with other molecules used in liposome targeting (54) , no covalent linkage or lipid tail seems to be required to retain CTT on the liposome surface under these conditions. In fact, we found that a synthetic CTT peptide containing a palmitate tail at its NH2 terminus was as active as CTT in liposome targeting (data not shown). However, the cyclic structure (i.e., an intact disulfide bond) is important (1) for gelatinase inhibition by the peptide and its ability to target liposomes into cells. The CTT-liposome complexes may not only be suited for delivery of cancer drugs but could also be used as gene expression vectors. It may also be possible that small peptides do not provoke immunological reaction to the same extent as do antibodies that are coupled to liposomes. Additional studies are in progress in our laboratories so as to investigate the utility of the properties of CTT in liposome targeting in vivo.


   ACKNOWLEDGMENTS
 
We thank Carmela Kantor and Tuula Nurminen (Department of Biosciences, Division of Biochemistry, University of Helsinki) for peptide synthesis; Birgitta Rantala and Kaija Niva for technical assistance; and Juha Holopainen and Arimatti Jutila for constructive criticism on the data and the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Academy of Finland, Finnish Cultural Fund, Finnish Cancer Society, and Tekes. Back

2 To whom requests for reprints should be addressed, at Department of Medical Chemistry, Institute of Biomedicine, P. O. Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Helsinki, Finland. Phone: 358-9-19125400; Fax: 358-9-19125600; E-mail: Paavo.Kinnunen{at}Helsinki.Fi Back

3 The abbreviations used are: MMP, matrix metalloproteinase; CLP, CLPGHWGFPSC; CTT, CTTHWGFTLC; CWL, CWLTFTHGTC; EGF, epidermal growth factor; eggPC, egg yolk phosphatidylcholine; LUV, large unilamellar vesicle; NBD-PE, 1-acyl-2-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl 1-sn-glycero-3-phospho-ethanolamine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; DPPRho, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamino-thiocarbamoyl-N-6-tetramethylrhodamine; RFI, relative fluorescence intensity; STT, STTHWGFTLS; TIMP-2, tissue inhibitor of metalloproteinase-2; TPA, phorbol ester; EthD-1,5,5'-[1,2-ethanediylbis(imino-3,1-propanediyl)]bis(3,8-diamino-6-phenyl)-dichloride, dihydrochloride; CHO, Chinese hamster ovary. Back

Received 4/26/00. Accepted 3/15/01.


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 DISCUSSION
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