Matrix Metalloproteinase-Assisted Triggered Release of Liposomal

Dec 14, 2007 - occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the t...
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Bioconjugate Chem. 2008, 19, 57–64

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Matrix Metalloproteinase-Assisted Triggered Release of Liposomal Contents Nihar Sarkar,† Jayati Banerjee,† Andrea J. Hanson,† Adekunle I. Elegbede,† Theresa Rosendahl,† Aaron B. Krueger,‡ Abir L. Banerjee,‡ Shakila Tobwala,‡ Rongying Wang,§ Xiaoning Lu,§ Sanku Mallik,*,† and D. K. Srivastava*,‡ Department of Pharmaceutical Sciences and Department of Chemistry, Biochemistry, and Molecular Biology, North Dakota State University, Fargo, North Dakota 58105, and Proteomics Core Facility, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202. Received March 8, 2007; Revised Manuscript Received September 25, 2007

We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically “uncorked” by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal “uncorking” and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site. The mechanistic details underlying the formulations of liposomes and their enzyme-selective “uncorking” and content release are discussed. Arguments are presented that such liposomes can be fine-tuned to serve as the drug delivery vehicles for the detection and treatment of various human diseases, which occur due to the overexpression of a variety of pathogenic matrix metalloproteinases.

INTRODUCTION Of the four major classes of extracellular matrix degrading enzymes (viz., cysteine proteases, aspartic proteases, serine proteases, and metalloproteases,), matrix metalloproteases (MMPs) have been implicated in several diseases (1, 2). On the basis of the structural features (including the amino acid sequences, domain organizations, etc.), 26 different types of MMPs have been recognized in human tissues, which fall into six major classes: (i) collagenases, (ii) gelatinases, (iii) stromelysins and stromelysin-like MMPs, (iv) matrilysins, (v) membrane-type MMPs, and (vi) other MMPs (viz., MMP-20, MMP-23, and MMP-28) (3). Although many of these MMPs have been implicated in different types of human diseases, MMP-2 and MMP-9 have been widely recognized to be involved in the progression and metastasis of most of the human tumors. They have been found to be overexpressed in breast tumors (4), colorectal tumors (5), lung tumors (6), prostate tumors (7), pancreatic tumors (8), and ovarian tumors (9). In view of their involvement in different human diseases, there have been major efforts to design MMPselective inhibitors as therapeutic agents and develop strategies for delivering them to their target sites (10). Of the different approaches of delivering drugs (e.g., via liposomes, polymers, microspheres, antibody-drug conjugates etc.), the liposome-assisted drug delivery systems offer several advantages. Presently, there are about 13 liposome-mediated drug delivery systems approved for treating a variety of human diseases (e.g., breast cancer, ovarian cancer, meningitis, fungal infections, leukemia, etc.), and about 30 additional systems for delivering small molecule drugs, DNA fragments, diagnostic compounds, and so forth, are currently at different stages of clinical trials (11, 12). Hence, the selective targeting of liposomes is of significant importance to ensure the delivery * [email protected]; [email protected], Tel: 701-2317888; Fax: 701-231-8333. † Department of Pharmaceutical Sciences. ‡ Department of Chemistry, Biochemistry, and Molecular Biology. § Proteomics Core Facility.

of therapeutic “loads” to the diseased tissue sites. This feature has been considerably optimized by attaching cell surfacespecific antibodies (13), peptides (14), and small molecule agonists/antagonists of the cell surface receptors (15) to differently formulated liposomes. Usually, liposomes slowly deliver their contents via fusion with the cell membranes, micropinocytosis, endocytosis, or simply by slow dissociation and passive diffusion of drugs from the liposomal lumen to the interior of the affected cells (11, 12). In order to accelerate the drug delivery process, liposomes are often “armed” with triggering devices. Some of the “triggering” agents include change in pH (16), mechanical stress (17), metal ions (18), temperature (19), light (20), enzymes (21), and so forth. Hence, both recognition of the diseased tissues and triggered release of the contents are the desirable features of the liposome-assisted drug delivery system. As will be noted in the following sections, both of these features are intrinsically built in our uniquely formulated liposomes. In addition, these liposomes can be “uncorked” in the vicinity of the cancerous tissues, which express high amounts of MMP-9. Out of about 25 000 papers (listed in PubMed) and 600 patents (listed at the USPTO site) on liposomes, there are only a handful of papers (less than 15) on the triggered release of liposomal contents by enzymes. Some of the enzymes utilized for cleaving liposomes and releasing their contents include elastase, alkaline phosphatase, trypsin, and phospholipase A2 (21). Herein, we report the design of triple helical peptides and their lipopeptide derivatives, incorporation of those lipopeptides into liposomes (such that the triple helical peptides protrude from the surface of the liposomes for facile cleavage), and MMP-9-assisted “uncorking” of the liposomes and release of their contents. We believe the overall strategy can be fine-tuned to diagnose and treat human diseases which occur due to overexpression of pathogenic matrix metalloproteinases, such as MMP-9 (22).

EXPERIMENTAL PROCEDURES Synthesis of the Peptides. The peptides were synthesized on a Rainin Symphony Quartet automatic peptide synthesizer,

10.1021/bc070081p CCC: $40.75  2008 American Chemical Society Published on Web 12/14/2007

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using standard Fmoc-protected amino acids and HBTU-HOBT as the coupling reagents. The commercially available CLEAR acid resin was used as the solid support. Each coupling step took 3 h and was repeated twice with 5-fold excess of reagents. Cleavage was performed for 3 h using a cocktail of CF3CO2H, anisole, and water (95%/2.5%/2.5%). The crude peptides were precipitated in ice-cold ether and purified by a semipreparatory RP-HPLC, using a linear gradient of 0–70% acetonitrile in water over 40 min. Each solvent contained 0.1% trifluoroacetic acid. The purified peptides were characterized by the CD and mass spectroscopy (MALDI-TOF). Conditions for Analytical HPLC. Vydac C18 analytical HPLC column (238TP5415); eluant, linear gradient of 0–70% acetonitrile in water over 40 min; both solvents contained 0.1% CF3CO2H; flow rate, 1.5 mL/min. Conditions for Semipreparatory HPLC. Vydac C18 HPLC column (238TP152022); eluant, linear gradient of 0–70% acetonitrile in water over 40 min; both solvents contained 0.1% CF3CO2H; flow rate, 5 mL/min. Synthesis of the Lipopeptides. After the completion of the peptide synthesis, the N-terminal Fmoc group was removed from the peptide. Conjugation with stearic acid was performed using the same procedure as the amino acid coupling with 5-fold excess of reagents. A shaker was used for better mixing of reagents. Cleavage conditions were the same as that for the peptides. The crude lipopeptides were purified by RP-HPLC, employing a Vydac semipreparatory diphenyl column (RP 219TP510). Analytical HPLC Conditions. Vydac analytical HPLC column (219TP5415); eluant, linear gradient of 0–70% acetonitrile in water over 40 min; both solvents contained 0.1% CF3CO2H; flow rate, 1.5 mL/min. Semipreparatory HPLC Conditions. Vydac HPLC column (219TP510); eluant, linear gradient of 0–70% acetonitrile in water over 40 min; both solvents contained 0.1% CF3CO2H; flow rate, 5 mL/min. The mass spectral data for the reported peptides and the lipopeptides are provided in Supporting Information. Circular Dichroism Spectroscopy. CD spectra were recorded on a Jasco J-815 instrument using a cell of 0.2 mm path length. The concentration of the peptides or lipopeptides was 1 mg/mL in 10 mM phosphate buffer, pH ) 4.0. The solutions were stored for 12 h at 4 °C before recording the spectra. For temperature-dependent CD spectra, the sample was equilibrated for 15 min at each temperature before recording the spectra. Cloning, Expression, and Purification of MMP-9. The catalytic and fibronectin domains (truncating the hemopexin domains from the full-length enzymes) of human MMP-9 were cloned in pET20b vector (Novagen), and the enzyme was overexpressed in BL21(DE3) Escherichia coli cells. The expressed proteins were primarily recovered from the inclusion bodies. The purified MMP-9 showed a single band on SDS gel electrophoresis. The yield from 1 L of bacterial culture was in the range 30–80 mg. The experimental details of the cloning, expression, and purification of MMP-9 will be published elsewhere. Peptide and Lipopeptide Cleavage Studies with MMP-9 and Trypsin. For the cleavage studies, the conditions are: [peptide] or [lipopeptide] ) 1 mg/mL in 25 mM HEPES buffer, pH ) 8.0, containing 10 mM CaCl2; [MMP-9] ) 5 nM, 25 °C. At different time intervals, 50 µL samples were withdrawn and 2 µL of CF3COOH was added to stop the reaction. The products were analyzed by RP-HPLC, using the same elution condition as employed for the analysis of the peptides and lipopeptides. Liposomal Formulation. MMPP_4HFA (30 mol %) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholime, 70 mol %, total lipid concentration of 1 mg/mL) were dissolved in CHCl3. A thin film was prepared by evaporating the solvent using a rotary evaporator. The film was placed under high

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vacuum for 12 h. The film was then hydrated with 100 mM 5-carboxyfluorescein solution (prepared in 25 mM in HEPES buffer, 10 mM CaCl2 at pH ) 8.0) for 1 h at 60 °C followed by sonication for another hour at 60 °C. Nonencapsulated dye was separated from liposomes via gel filtration chromatography with a Sephadex G75 column. Before loading the crude liposomal preparation on the column, the osmolarity of the elution buffer (with same composition) was adjusted by the addition of solid NaCl. Kinetics of Release of Carboxyfluorescein from Liposomes. MMP-9 (10 µL of 200 µM solution) was added to a 2 mL solution of liposome in 25 mM HEPES buffer, pH 8.0, containing 10 mM CaCl2 at 25 °C. The emission spectra of the control and liposomes with MMP-9 were measured. The emission intensity at 518 nm (excitation: 480 nm) was monitored as a function of time for 5 h. The experimental condition for the leakage studies with trypsin was the same as that for MMP-9.

RESULTS It is known that incorporation of Gly Pro-Hyp repeats in polypeptide chains tend to form the triple-helical collagenmimetic peptides (23). The magnitude of such helicity increases with either attachment of alkyl groups at the N-terminus (24) or introduction of cysteine knots (25), or metal ions (26). To incorporate collagen-mimetic triple helical peptides on the surface of liposomes for their recognition by MMP-9 and subsequent cleavage, we synthesized a few such peptides and conjugated them with stearic acid via their N-terminal NH2 groups. Four criteria were taken into consideration for syntheses of such lipopeptides: (i) the peptides should exhibit the triple helical conformations, (ii) the peptides should contain the cleavage site for MMP-9, (iii) the peptides should be resistant to the cleavage by general proteases, (iv) the triple helical peptides must exhibit propensity to undergo facile unwinding to be effectively cleaved by MMP-9. With these criteria in mind, we synthesized a series of lipopeptide conjugates and investigated their physicochemical properties. Although the natural substrate for MMP-9 is the type IV collagen (which is cleaved between Gly439-Val440 bond), the enzyme exhibits overlapping substrate specificity for a variety of synthetic (collagen mimetic) peptide substrates (27, 28). Usually, the enzyme cleaves between G and X (where X ) Leu, Ile, or Val) of a peptide sequence GPQGXAGQR. In view of the amino acid sequence around Gly904 of type I collagen, as well as GPO or GPP repeats (responsible for forming the triple helical units), we synthesized the following peptides: MMPP3_H: H2N-G904PQGLAGQRGIVGLOG919-COOH MMPP4_H: H2N-G904PQGIAGQR(GPO)4GG-COOH MMPP4_GPP: H2N-G904PQGLAGQR(GPP)4GG-COOH MMPP5_H: H2N-G904PQGLAGQR(GPO)4GG-COOH MMPP7_H: H2N-G(GPO)4GLAGQR(GPO)4GG-COOH MMPP8 (control): H2N-G904PQGAAGQRGIVGLOG919COOH. Whereas these peptides are known to be efficiently cleaved by collagenases (MMP-1, -8, and -13), they are also cleaved (albeit somewhat sluggishly) by gelatinases (MMP-2 and -9) (28, 29), and thus they are ideally suited for investigating the MMP-9-assisted cleavage reaction on longer time scales. We undertook the standard Fmoc-based, solid-phase synthetic protocol for synthesizing the above peptides, and subsequently purified those by the HPLC method. Of these, MMPP8 was neither expected to be cleaved by MMP-9 (due to the lack of the cleavage site) nor had the potential to form the triple helix (due to the lack of the GPO or GPP repeats). On the other hand, although MMPP3_H could not form the triple helical structure (due to the lack of the GPO or GPP repeats), it contained the

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Bioconjugate Chem., Vol. 19, No. 1, 2008 59 Table 1. Rpn Values and Melting Temperatures (Tm) of Synthetic Peptides and Lipopeptides

Figure 1. Circular dichroic spectra of MMPP3_H (inset), MMPP4_H and MMPP4_HFA in 10 mM phosphate buffer, pH ) 4.0 at 4 °C. The concentration of each peptide/lipopeptide was 1 mg/mL. The solutions were stored at 4 °C for 12 h before recording the spectra.

cleavage site for MMP-9. The GPP repeats in MMPP4_GPP and the presence of the GPO repeats on both sides of the cleavable sequence in MMPP7_H were utilized to ascertain their potentials to form the triple helices, as well as the feasibility of unwinding during the MMP-9-dependent cleavage reaction. The N-termini of all these peptides were conjugated with stearic acids using the HBTU/HOBt coupling agents. The corresponding lipopeptides (identified by the suffix “FA”, to denote the fatty acid conjugation, after the name of the parent peptides) were subjected to final purification by HPLC, followed by their characterizations by CD and mass spectroscopy. Figure 1 shows the representative CD spectra of MMPP3_H, MMPP4_H and its fatty acid conjugate MMPP4_HFA. Note that all these peptides are characterized by negative elliptical bands at 196–200 nm and positive elliptical bands at 220–223 nm regions. However, the latter band is miniscule (or nonexistent) in the case of MMPP3_H (Figure 1, inset), suggesting the lack of the triple helical structure in its sequence. This was not unexpected, since it contained neither the GPO nor GPP repeat units. In contrast, both MMPP4_H and MMPP4_HFA exhibited pronounced ellipticities in the 220–223 nm regions, suggesting their ordered helical structures (Figure 1). The ratio of θ198 to θ225 (referred to as the Rpn value) (29) for MMPP4_H and MMPP4_HFA was found to be equal to 0.04 and 0.1, respectively. Given that the Rpn value of naturally occurring “collagen” is equal to 0.13 (30), it appears evident that the addition of a fatty acid chain to MMPP4_H (forming MMPP4_HFA) considerably enhances the magnitude of the triple helicity in the above peptide. We recorded the spectra of all the synthetic peptides and their fatty acid conjugates (Supporting Information) and calculated their Rpn values (Table 1). The data of Table 1 reveal that the magnitude of the triple helicity of all peptides containing GPO (and to a lesser extent containing GPP) repeats increases upon converting them to their fatty acid derivatives. As expected, the control peptide MMPP8 did not show any evidence of the triple helical structure. These data are consistent with the previous studies of Fields and his collaborators on the alkyl group assisted formation of the triple helical structures in collagen-mimetic peptides (24). To determine the stability of our synthetic collagen-mimetic peptides, we subjected them to thermal denaturation. We incubated each peptide at different temperatures for 15 min and then recorded their CD spectra. Figure 2 shows a representative set of CD spectra of MMPP4_H (Figure 2A) and MMPP4_HFA (Figure 2B) as a function of temperature. The

peptides/ lipopeptides

Rpn

Tm (°C)

MMPP3_H MMPP3_HFA MMPP4_H MMPP4_HFA MMPP4_GPP MMPP4_GPPFA MMPP5_H MMPP5_HFA MMPP7_H MMPP7_HFA MMPP8 MMPP8_FA

0.04 0.1 0.04 0.05 0.099 0.06 0.08 -

57 34 37 75 42 55 -

temperature-dependent spectral data of both these peptides exhibit a decrease in the magnitude of the negative and positive elliptical peaks, with isosbestic points at 210 and 213 nm, respectively. This feature is consistent with a one-step transition between triple helices and their monomeric forms. The insets of Figure 2 show the positive elliptical peaks at 225 nm as a function of temperature. The solid lines are the best fits of the data according to the conventional equation (24) for determining the Tm value from the temperature-dependent spectral changes. As apparent from the data of Figure 2A (inset), the Tm value of MMPP4_H is not easily resolvable, and its magnitude appears to be smaller than 57 °C for MMPP4_HFA peptide (Figure 2B, inset). Hence, besides enhancing the extent of triple helicity, the fatty acid conjugation with MMPP4_H increases its stability. Identical experiments were performed with other synthetic collagenmimetic peptides and their fatty acid conjugates (Supporting Information). It should be mentioned that, although the thermal transition of most of the peptides/lipopeptides conformed to the single isosbestic point, MMPP4_GPPFA exhibited at least two isosbestic points (Supporting Information). The origin of the latter features is not clear at this point. A cumulative account of the spectral and thermal transition data of Figures 1 and 2 for all peptides and lipopeptides is presented in Table 1. An inspection of spectral and thermodynamic parameters of different peptides/lipopeptides revealed that MMPP4_HFA would be the best-suited lipopeptide for formulating liposomes and investigating their uncorking by MMP-9. Hence, all subsequent experiments were performed with MMPP4_H and MMPP4_HFA pair. Since the cleavage and the liposomal leakage studies are performed at pH ) 8.0, we recorded the CD spectrum of the lipopeptide MMPP4_HFA at that pH. The CD spectrum indicated that MMPP4_HFA retains its triple helical conformation at pH ) 8.0 (Rpn ) 0.12) with a melting temperature of 42 °C (Figure S3, Supporting Information). To determine whether or not MMPP4_H and MMPP4_HFA would be efficiently cleaved by MMP-9, we incubated individual peptides (5 µM) with 5 nM MMP-9 in 25 mM HEPES buffer, pH ) 8.0. At different time intervals, 50 µL aliquots were withdrawn and transferred to a vial containing 2 µL of trifluoroacetic acid (TFA). The latter was used to denature the enzyme and stop further cleavage of the peptides. The aliquots collected at different time intervals were subjected to the HPLC analysis using a C18 Vydac analytical column and monitoring the elution profile at 214 nm. Figure 3A,B shows the time course of the HPLC profiles for the cleavage of MMPP4_H and MMPP4_HFA, respectively, by MMP-9. From the HPLC profiles of Figure 3A, it is evident that, at zero time, MMPP4_H elutes as a single (nearly symmetrical) peak at 15 min. Upon cleavage for 5 min, the above peak diminishes, and a new peak emerges (due to the formation of one of the cleaved products exhibiting high absorption at 214 nm) at 13 min. The absence

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Figure 2. Temperature-dependent CD spectra for MMPP4_H (A) and MMPP4_HFA (B) are shown. The insets show the melting curves obtained by plotting the peak intensities at 225 nm as a function of temperature. The solid lines in the insets are the fitted curves. The experimental conditions are the same as those of Figure 1.

Figure 3. HPLC elution profiles for the cleavage of MMPP4_H (A) and MMPP4_HFA (B). The cleavage reactions were performed using 5 µM peptide/lipopeptide and 5 nM MMP-9 in 25 mM HEPES buffer, pH ) 8.0, 25 °C.

of the second peak (expected to be formed upon treatment with MMP-9) on the HPLC profile is presumably due to its shorter chain length, showing low absorption at 214 nm. As the time of cleavage reaction progresses, the peak at 15 min decreases in magnitude and the peak at 13 min increases. This pattern is suggestive of the MMP-9-assisted cleavage of the peptides. Due to the presence of fatty acid chain in MMPP4_HFA, the HPLC cleavage peaks were significantly separated, with reactant and products eluting at 27 and 14 min, respectively. As observed for the cleavage of MMPP4_H, the diminution of the reactant HPLC peak at 27 min proceeded concomitant with the increase in the product peak at 14 min. Qualitatively, it appeared that the MMPP4_H was being cleaved about 4-fold more efficiently than its fatty acid conjugate, MMPP4_HFA, by the same amount of MMP-9. Given that the magnitude of triple helicity is more pronounced with MMPP4_HFA (Rpn ) 0.1) compared to MMPP4_H (0.04), it is tempting to speculate that the MMP-9-assisted cleavage of the collagenmimetic peptide requires unwinding of the triple helix. Such a mechanistic feature was also envisaged by Fields (31). As a hindsight control, we probed whether or not a general proteolytic enzyme, such as trypsin, can cleave either MMPP4_H or MMPP4_HFA or both. Trypsin was chosen because the above peptides contained the Arg-Gly peptide bond, which is recognized and cleaved by the enzyme. We observed that, whereas trypsin could efficiently cleave the above bond in the case of MMPP4_H peptide, it failed to cleave the same

bond in MMPP4_HFA lipopeptide (at least up to 2 h of incubation; data not shown). Clearly, the trypsin cleavable bond is not accessible to the enzyme in the triple helical conformation of the lipopeptide. Having established the cleavage of the collagen-mimetic triple helical MMPP4_HFA by MMP-9, we proceeded to formulate liposomes using the above lipopeptide as minor components (30 mol %) and 1-palmotoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as the major component (70 mol %). While preparing liposomes, a self-quenching fluorescent dye (carboxyfluorescein) was encapsulated (32). The free dye was separated from the liposome-encapsulated by the Sephadex G-75 gel filtration column chromatography. The dye-encapsulated liposomes thus prepared showed a fairly uniform distribution of their size on TEM, and their average diameter can be determined as being equal to 50 nm (Supporting Information). The fluorescence emission intensity of liposome encapsulated carboxyfluorescein (λex ) 480 nm, λem ) 518 nm) remains quenched (due to the local concentration effect) until it is released to the surrounding medium (32). This feature serves as a prototype for monitoring the release of the liposomal content under different experimental conditions. We determined the influence of MMP-9 on cleavage of the MMPP4_HFA lipopeptide incorporated in the liposomes, and their subsequent destabilization and release of the entrapped carboxyfluorescein. During these experiments, we incubated 2 mL solution of liposomes (100 µg lipids) with 1 µM MMP-9 in 25 mM HEPES

MMP-9-Assisted Release of Liposomal Contents

Figure 4. Time courses of release of carboxyfluorescein from liposomes under different conditions. Trace 1: in the presence of 1 µM MMP-9; the solid smooth red line is the best fit of the data according to eq 2 with k1 and k2 values of 0.018 and 0.014 min-1, respectively. Trace 2: in the absence of MMP-9 (control). Trace 3: in the presence of 1 µM trypsin. The blue circles indicate the release profile from control liposomes (formulated without MMPP4_HFA) in the presence of 1 µM MMP-9. The solid lines of traces 2 and 3 are linear regression analyses of the data.

buffer, pH ) 8.0, containing 10 mM CaCl2, and monitored the time course of the increase in the fluorescence emission intensity at 518 nm (λex ) 480). Figure 4, trace 1, shows the timedependent profile for the release of carboxyfluorescein under the above experimental condition. To ensure that the release of carboxyfluorescein from liposome was due to the cleavage of the collagen-mimetic lipopeptide, we performed a control experiment exactly under the above condition, but without MMP-9 (Figure 4, trace 2). Note that the time-dependent release of the dye in the absence of MMP-9 (trace 2) is far less pronounced than that observed in the presence of the enzyme (trace 1). To calculate the percent release of the dye during the above experiments, the liposomes were completely disrupted with a 5% aqueous solution of a detergent, Triton-X, and the total increase in fluorescence was determined (taken as a measure of 100% release of the dye from the liposomes). Given these data, we propose that, due to cleavage of the surfaceexposed collagen-mimetic triple helical peptide by MMP-9, the lipid domains of the liposomes are destabilized, resulting in the release of the encapsulated fluorescent dye. The overall process is cumulatively referred to as the liposomal “uncorking”. If the liposomes do not contain any MMPP4_HFA, the enzyme MMP-9 does not release the dye to any significant extent (Figure 4, blue circles). We already demonstrated that, despite the presence of the trypsin cleavage site in MMPP4_HFA, the enzyme does not cleave the latter lipopeptide. If this property remained unchanged upon incorporation of MMPP4_HFA into liposomes, trypsin would not be able to “uncork” the liposomes. This expectation was clearly substantiated when we incubated the MMPP4_HFAcontaining liposomes with 1 µM trypsin, precisely under the experimental conditions of trace 1 of Figure 4. As shown by trace 3 of Figure 4, the time course of leakage of the fluorescent dye matches that of the control experiment (performed in the absence of any enzyme), and it is significantly slower than that observed in the presence of MMP-9 (trace 1, Figure 4). As an additional control, we prepared the liposomes incorporating the stearic acid conjugate of the peptide MMPP8 (i.e., MMPP8_FA) encapsulating carboxyfluorescein. In the presence of MMP-9 (1 µM), these liposomes showed a release profile very similar to trace 3 in Figure 4 (data not shown). The lipopeptide MMPP8_FA does not contain the cleavage site for the enzyme MMP-9; hence, it is not surprising that MMP-9

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does not release the encapsulated dye from the liposomes incorporating the lipopeptide MMPP8_FA. One apparent feature of the MMP-9-assisted uncorking of liposomes is the presence of a lag phase. A simple mechanistic explanation for the prevalence of such a lag phase is the involvement of at least one intermediary step during the overall uncorking process. However, even if one or multiple intermediary steps predominate during the course of liposome uncorking, the observed signal is given only by the carboxyfluorescein release step. For example, if the above liposome is cleaved by MMP-9, but its content is not released until the lipid domains are reorganized, the overall kinetic profile would involve a finite lag phase similar to that observed in Figure 4. Assuming that the MMP-9-assisted uncorking adhered to a two-step process, we analyzed the kinetic profile of Figure 4 (trace 1) by a twostep mechanism (see Discussion). The solid smooth (red) line is the best fit of the data for the k1 and k2 values of 0.018 ( 0.0034 and 0.014 ( 0.0016 min-1, respectively. However, it should be noted that the fitted line does not precisely correspond to the experimental data (broken black line, trace 1). Clearly, the overall uncorking process is more complex than that explainable in light of the two-step mechanism (see Discussion).

DISCUSSION The formulation of MMP-9-cleavable liposomes required synthesis of collagen-mimetic peptides, which could provide the cleavage sites for the enzyme. Of the different lipopeptides (synthesized on the basis of the amino acid sequences present around the cleavable site of type I collagen), MMPP4_HFA was found to be the best-suited for formulating such liposomes. The other lipopeptides either did not form adequate triple helical structures, comparable to those found in the natural collagen (e.g., MMPP3_HFA, MMPP4_GPPFA, MMPP8_HFA), or they formed triple helices that were too compact as evidenced by their high Tm values (e.g., MMPP5_HFA), cleaved somewhat sluggishly by MMP-9 (MMPP7_HFA), or cleaved nonspecifically, such as by trypsin (MMPP7_HFA). It should be mentioned that, although MMPP4_H contained the trypsincleavage site (and it was cleaved by the enzyme in its native/ unconjugated state), its stearic acid derivative (i.e., MMPP4_HFA) was not cleaved by trypsin. Clearly, the trypsin cleavage site of the original (monomeric) peptide (MMPP4_H) is masked upon formation of the triple helical structure in MMPP4_HFA. Whether or not the oligomerization of polypeptide units in triple helical peptides incorporates selectivity for matrix metalloproteinases, and obviates their cleavage by general proteases (e.g., trypsin, chymotrypsin, etc.), remains to be investigated. However, our experimental data provide clues as to why cellular matrices are constituted of fibrillar proteins, and they are specifically cleaved by matrix metalloproteinases. The fact that a small segment of collagen-mimetic peptide (e.g., MMPP3_H) is devoid of the triple helical structure, whereas the native collagen is predominantly triple helical in nature, implies that the origin of the triple helicity lies in the cooperative interaction among the extended polypeptide chains. The latter feature is augmented by the presence of the GPO and GPP repeats in the polypeptide chains. The cooperative interaction among “shorter” chain containing peptides appears to be significantly enhanced upon attachment of C18 hydrophobic tail at the N-terminal end (24). This is apparent from the data of Figure 1; the Rpn value of MMPP4_H is increased from 0.04 to 0.1 upon conjugation with stearic acid. The question arises whether the Rpn value of 0.04 in MMPP4_H is simply due to its preponderance in the form of polyproline II helix (33) or its ability to form a partial triple helical structure. In attempting to answer this question, we realized that the peptide containing the GPP (instead of GPO) repeats (viz.,

62 Bioconjugate Chem., Vol. 19, No. 1, 2008 Scheme 1. Proposed Thermodynamic Model of the Monomer-Trimer Equilibria of MMPP4_HFA Lipopeptide

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increase in the fluorescence intensity involved a finite lag phase, and the signal of measurement came from the release of carboxyfluorescein to the exterior environment, a minimal twostep model of uncorking was envisaged (eq 1). k1

k2

L - F 98 L * F 98 L * + F

MMPP4_HGPP) does not contain any discernible positive elliptical peak at 225 nm. Since the presence of the GPO repeats in the peptide sequence facilitates the formation of the triple helical structure, it follows that MMPP4_H exists in an equilibrium mixture of the monomeric and trimeric (i.e., triple helical) forms. This equilibrium is favored toward the trimeric form upon conjugation with the stearic acid as depicted by the model of Scheme 1. Consistent with the model of Scheme 1, the temperaturedependent CD spectra of MMPP4_H and MMPP4_HFA (Figure 2) conform to single isosbestic points. As the temperature increases, the magnitude of elipticity at both 195 and 225 nm decreases, albeit by different amounts. This feature results in a continuous diminution in the Rpn value as a function of increasing temperature, presumably because of the conversion of the triple helical form (trimer) form of MMPP4_HFA to the monomeric form. The observation that MMPP4_H appears to be more efficiently cleaved by MMP-9 than MMPP4_HFA implies that the conjugation of stearic acid impairs the catalytic activity of the enzyme. We believe the origin of the above disparity lies in the preference of MMP-9 to cleave the monomeric rather than triple helical peptides. This is consistent with the postulate that MMP-9 unwinds the triple helical peptides prior to cleaving them (31). Given this, we surmise that the sluggish rate of cleavage of MMPP4_HFA (Vis a Vis MMPP4_H) by MMP-9 is due to the rate-limiting unwinding of the triple helical peptide to its monomeric form. The question arises as to why the triple helical form of the peptide is not unwound to produce the monomeric unit prior to their cleavage by trypsin. The most plausible answer to this question is that the unwinding of the triple helix requires assistance from specialized proteases, such as MMP-9 (which contain noncatalytic fibronectin domain), and such functions are not accomplished by general proteases such as trypsin. A marked success in our overall endeavor has been the demonstration that the lipopeptide, MMPP4_HFA, could be easily incorporated in the liposomes, and the latter are selectively “uncorked” by MMP-9 but not by trypsin. On the basis of the literature (32), it is expected that the C16-hydrocarbon moiety of the lipopeptide MMPP4_HFA will be embedded in the lipid bilayer of the liposomes. The triple helical peptide portion of the molecules will protrude outside the lipid bilayer. The amino acids GPQ may act as a spacer, making the MMP-9 cleavage site of the peptide accessible to the enzyme. Since the cleavage of the triple helical peptides destabilizes the lipid domains, their contents are released. Due to ease in performing the liposomal uncorking experiments, we chose a self-quenching fluorescent dye, carboxyfluorescein. As long as the dye remains confined inside the liposomal lumen, its fluorescence remains quenched (33). As soon as the dye leaks out from the liposomes, its fluorescence starts increasing. The time course of the increase in fluorescence emission intensity at 518 (Figure 4) is the sum of a sequence of microscopic events associated with the overall uncorking process. Hence, it is not surprising that the overall kinetic profile is complex (Figure 4, trace 1). However, since the kinetics of

(1)

Where L and F represent liposomes and carboxyfluorescein, respectively. The first step involves the cleavage and destabilization of the liposomes. The intermediary species L*-F is representative of the cleaved liposomes, but fluorescence dye still remains confined in the liposomal lumen. The second step is release of the fluorescence dye with concomitant increase in the fluorescence emission intensity at 518 nm. For this two irreversible step model, the time course of formation of F can be given by eq 2 (34).

[ (

F ) [L-F] 1 +

1 k1 - k2

)(

]

k2e-kt - k2e-kt)

(2)

The solid, smooth line of trace 1 (Figure 4) is the best fit of the data according to eq 2, with k1 and k2 being equal to 0.018 ( 0.0034 min-1 and 0.014 ( 0.0016 min-1, respectively. However, since the fitted line (red) somewhat deviates from the experimental data (broken black lines), it appears that the sequence of events of the MMP-9-assisted uncorking is more complex than that conceivable in light of the two-step model. This is not unexpected, since even after the cleavage of the triple helical peptides, a series of microscopic steps may intervene prior to the onset of the final step of the dye release. The lipopeptide MMPP4_HFA contains one fatty acid, and this may not form tight domain boundaries with the major lipid of the liposomes, POPC (35). The 10% release of the dye in the absence of MMP-9 (Figure 4, trace 2) may arise from these domain boundaries. We have observed that, in buffer solutions (pH ) 8.0), the recombinant MMP-9 self-hydrolyzes and inactivates itself within 3 h. This self-hydrolysis is one of the possible reasons for not releasing all of the liposomal contents. In order to test this hypothesis, we repeated the liposomal release experiments under the conditions as described previously. We observed that the rate of release of the encapsulated carboxyfluorescein decreases considerably after 130 min (54% release, as indicated in Figure 4). However, the addition of a second aliquot of MMP-9 (10 µL of 200 µM solution) restarted the release of carboxyfluorescein from the liposomes. Cumulatively, 94% of the encapsulated dye was released in 6 h (Figure 5). We are currently in the process of fine-tuning the molecular mechanism of liposomal uncorking and its content release, and we will report on these findings subsequently. In conclusion, we have provided the first detailed studies on the formulation of liposomes, which harbor the MMP-9cleavable collagen-mimetic peptides on their surface. The cleavage of the above peptides by MMP-9 results in destabilization of the lipid domains of the liposomes, leading to the release of their encapsulated contents (referred to as “uncorking”). Since MMP-9 is invariably expressed in most of the cancerous tissues, our liposomal formulation will find unique application as a drug delivery vehicle not only for inhibiting MMP-9, but for all other pathogenic enzymes/proteins which are expressed at the target sites. When liposomes will be encapsulated with an MMP-9specific inhibitor, the triple helical peptides will serve as the “bait” for the enzyme, and the enzyme will essentially “commit suicide” once it “uncorks” the liposomes and releases its own inhibitor. Once the enzyme is inhibited, it will lose its potency to further uncork the liposomes. In this way, our overall approach has the potential to serve as a “self-adjusting” drug delivery system, where the amount of drug released will be

MMP-9-Assisted Release of Liposomal Contents

Figure 5. The increase in the fluorescence intensity due to the release of the liposome-encapsulated dye (carboxyfluorescein) is shown (λex ) 480 nm, λem ) 518 nm) in the presence of the recombinant human MMP-9 (blue trace, 1 µM enzyme, 25 mM HEPES buffer, pH ) 8.0, 20 °C) and in the absence of the enzyme (red trace). A second aliquot of MMP-9 was added after 150 min. In the presence of MMP9, 94% encapsulated carboxyfluorescein was released in 6 h.

dictated by the amount of the catalytically active enzyme present in its resident environment. On the other hand, if the liposomes are encapsulated with fluorescent or magnetic resonance contrast agents, the triggered release of their contents will find usage in imaging the diseased tissues (expressing MMP-9). Both these features of our uniquely designed liposomes are of prime importance in diagnosis and treatment of a wide variety of cancers in human patients.

ACKNOWLEDGMENT This research was supported by the NIH grant 1R01 CA113746 and NSF DMR-0705767 to S.M. and D.K.S. A.J.H. was supported by the NSF-EPSCoR award EPS-0447679. X.L. thanks the support of the proteomics core facility by NIH Grant P20 RR016741 from the INBRE Program of the NCRR. Supporting Information Available: Mass spectral data and CD spectra for the synthesized peptides and lipopeptides and TEM picture of the liposomes incorporating MMPP4_HFA. This material is available free of charge via the Internet at http:// pubs.acs.org/BC.

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