SEPTEMBER 2009 Volume 20, Number 9 Copyright 2009 by the American Chemical Society
REVIEWS Proteolysis: A Biological Process Adapted in Drug Delivery, Therapy, and Imaging Benedict Law*,† and Ching-Hsuan Tung‡ Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota 58105, and The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, Texas 77030. Received November 19, 2008; Revised Manuscript Received January 15, 2009
In many diseases, protease expressions are found deregulated when compared with them at the healthy states. The unique ability to hydrolyze protein amide bonds has made those deregulated proteases attractive biological triggers in drug development. Proteolysis has been widely applied in pro-drug design to achieve favorable pharmacokinetics. Controlled drug delivery systems are also reported by incorporating protease-sensitive motifs onto bio-, inorganic-, or organic- materials. In addition, protease responsive molecular probes are developed for in vitro bioanalysis and in vivo diagnostic imaging. This review focuses on various proteolysis-dependent approaches to drug delivery, therapy, and imaging. References are selected to illustrate the concepts and demonstrate the potentials of these enzyme-responsive strategies.
INTRODUCTION Proteolysis is a simple hydrolytic process that separates two adjacent amino acid residues at the amide bond, aided by proteases (or proteinase). Without the catalytic assistance of protease, protein hydrolysis is a very slow process. Proteolytic activities are tightly regulated through various mechanisms. For example, intracellular proteolysis is regulated by segregation within organelles such as lysosomes. Some proteases are expressed as pro-enzymes, while others are complexed with * Benedict Law, Ph.D., Department of Pharmaceutical Sciences, 1401 Albrecht Blvd, Sudro Hall 116, North Dakota State University, Fargo, ND 58105. E-mail:
[email protected]. Ching H. Tung, Ph.D., Department of Radiology, The Methodist Hospital Research Institute, Weill Medical College of Cornell University, 6565 Fannin Street, #B5022 Houston, TX 77030. Email:
[email protected]. † North Dakota State University. ‡ Weill Cornell Medical College.
natural enzyme-inhibitors. Their enzymatic properties can be restored only when they are in demand. As disease initiated, misregulation of enzymatically active proteases or launching of new foreign proteases can occur, resulting in various host responses. Diseases can be controlled by stopping or slowing diseaseassociated abnormal proteolysis. Protease inhibitors have a long history as therapeutic agents. Synthetic inhibitors targeting various proteases have been developed and clinically used in treating a large variety of diseases (1). Cancer is a well-known example. Protease inhibitors against matrix metalloproteinases (MMP) have been proposed to control tumor growth; unfortunately, the outcomes were not as originally expected (2). Beyond cancer, protease inhibitors have been applied or proposed for treating diseases such as thrombosis (3), hypertension (4), diabetes (5), cognitive disorders (6), infectious diseases (7), and others. Clinically approved protease inhibitors have supported
10.1021/bc800500a CCC: $40.75 2009 American Chemical Society Published on Web 03/09/2009
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Table 1. Examples of Amino Acid and Peptide Caged Prodrugs That Are Activated by Their Target Proteasesa active drug metabolite Doxorubicin Methotrexate Methotrexate Amiloride Cyclopamine Doxorubicin
conjugated peptide-substrate L (13) A (20) Pyroglutamyl (21) LFGGGG (143) SSKYQ (144) BLAL (25) PLGL (32)
aFK (145) Fluorodeoxyuridine HSSKLQL (34) Gentamicin GfPRGFPAGG (70) Methotrexate Pyr (21) aFKK (146) Paclitaxel aFK, vLK (38) Thapsigargin HSSKLQL (35) Vinblastine Hyp-SS-Chg-QSSP (33)
target protease
disease
cathepsin B carboxypeptidase aminopeptidase enkephalinase PSA TOP MMP-2, MMP-9 plasmin
cancer cancer cancer ischemia cancer cancer cancer cancer
PSA thrombin PYA cathepsin, plasmin plasmin PSA PSA
cancer infection cancer arthritis cancer cancer cancer
a Note: The small letters in the peptide sequences indicate that particular amino acids are in D-configuration. All amino acid derivatives are shown by a group of three italics. Abbreviations: Chg, L-cyclohexylglycine; Hyp, trans-4-hydroxy-L-proline; PSA, prostate specific antigen; PYA, pyrogluatmate aminopeptidase; Pyr, L-pyroglutamic acid; TOP, thimet oligopeptidase.
the original hypothesis that active site blocking could relieve, correct, or slow disease progress. In contrast, since upregulated proteolysis is often seen during disease progression, instead of blocking its catalytic property, proteolysis has been proposed as a unique in vivo process to assist therapy and diagnosis. Numerous approaches based on proteolysis have been crafted to manage diseases; for example, prodrugs are designed to be activated by proteases, delivery systems are constructed to release drugs in a controllable manner, and molecular reporters are prepared to be protease sensitive for disease detection and imaging.
APPLICATIONS OF PROTEOLYSIS Protease-Activatable Prodrug. The rationale behind prodrug design is to improve bioactivity and/or bioavailability of a therapeutic agent. A prodrug generally has minimal pharmacological effect in its native form, but can be converted to its active pharmacophore when triggered in vivo. The concept of prodrugs is not new. For example, enalapril, an antihypertensive precursor of enalaprilat, was chemically modified to improve its absorption from the gastrointestinal tract (8), and capecitabine was designed to reduce the adverse effects of its cytotoxic metabolite, 5′-fluorouracil (FU) (9). Rational prodrug design requires an extensive knowledge of chemical entities that are likely to alter resulting pharmacokinetic properties. In addition, a profound understanding of disease molecular pathologies and their heterogeneities versus normal health states helps to identify potential activators. As mentioned, excessive proteolysis is involved in many diseases such as cancer, cardiovascular injury, and inflammation; thus, disease-associated proteases are attractive targets for pro-drug developments. In fact, numerous protease-activatable prodrugs have been reported (10, 11), while the majority have been developed for cancer treatment (12) (Table 1). Amino Acid Caged Prodrugs. To design a protease-activatable prodrug, a single amino acid or a dipeptide can simply be attached to a free drug via the formation of an amide bond; the resulting conjugate (prodrug) with significantly reduced potency can be hydrolyzed by selective aminopeptidase later to generate clinically effective metabolite (Figure 1A). L-Leucyl-doxorubicin is an early example developed in the 1980s (Figure 1B) (13). It is four times less toxic than its parent doxorubicin and has been shown to reduce cardiotoxicity in preclinical studies (14-16). Several studies of human breast, lung, and ovarian tumor
Figure 1. (A) Schematic presentation of an amino acid-drug conjugate activated by aminopeptidase. (B) The chemical structure of L-leucyldoxorubicin. Leucine is covalently conjugated to the aminoglycoside portion of doxorubicin (13). (C) The chemical structure of various 2-Rcarboxyl methotrexate derivatives (21).
xenografts demonstrated that L-leucyl-doxorubicin had not only a better therapeutic index, but also a greater efficacy than doxorubicin (17, 18). A high concentration of active metabolite doxorubicin was found in tumor cells, suggesting that the enzymatic cleavage of L-leucyl-doxorubicin takes place either on the surface inside of the tumor cells. Tissue peptidases such as cathepsin B have been proposed to be the responsible activators (19). Prodrugs with a similar strategy have also been described for methotrexate (20). Amino acids such as alanine, aspartic acid, and arginine were conjugated to the R-carboxyl of methotrexate via an amide linkage (Figure 1C). Ex vivo studies indicated that the resulting alanine-conjugate was 100 times less toxic than its parent drug. On the other hand, cytotoxicity was greatly enhanced by carboxypeptidase activation. Protease selectivity could be altered by modifying a different position on methotrexate (21). A series of 2-L-leucyl, 2-L-valyl, 2-L-isoleucyl, and 2-L-pyroglutamyl methotrexate conjugates were synthesized by attaching the corresponding amino acids to the NH2-group at the 2-position of the pteridine ring (21). Among all of these, 2-L-pyroglutamyl analogue was considered the best drug candidate, as it can be selectively activated by pyroglutamate aminopeptidase but is resistant to nonspecific enzymatic degradation in serum. Peptide Caged Prodrugs. Although the amino acid caging approach shows promise, the prepared prodrugs are not stable enough in circulation. With L-leucyl-doxorubicin as an example, pharmacokinetic studies have shown that leucyl-doxorubicin was rapidly converted into free doxorubicin in plasma compartment (19). Its superior therapeutic efficacy over doxorubicin is most likely due to the higher accumulation of this metabolite (free doxorubicin) in tumor than in normal tissue. To design a more stable prodrug, a tetrapeptide conjugate, BLAL-doxorubicin (B ) β-alanine), was synthesized (Figure 2) (22). The β-alanine at the N-terminus was incorporated to improve stability in blood. In vitro and in vivo studies with a similar prodrug analogue, N-(succinyl)-BLAL-doxorubicin (CPI-0004Na), have shown that extracellular activation is a multistep process (22-24): (1) The prodrug was first hydrolyzed extracellularly into AL-doxorubicin. This process was also proven to be the rate-limiting step; (2) AL-doxorubicin was subsequently cleaved into leucyldoxorubicin; and (3) it finally entered the cell and was converted into fully active doxorubicin. Thimet oligopeptidase (TOP) and
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Figure 2. Schematic presentation of a peptide-prodrug conjugate activated by multiple proteases. Prodrug acylated with a specific peptide substrate is cleaved by its target protease, and the remaining peptide-drug intermediates can be further digested by different aminopeptidases to release the free drug.
neprilysin (CD10) were identified as the responsible initial activators (25, 26). However, the choice between aminopeptidase and carboxypeptidase is limited; more sequence selectivity would be favored to achieve disease-specific prodrug activation. Using the same construct as shown in Figure 2, conjugating longer specific peptide substrates recognized by endopeptidases to an active pharmacophore has been done to obtain better protease selectivity. Peptide-caged prodrugs activated by plasmin (27-29), MMPs (30-32), and prostate-specific antigen (PSA) (33-36) have all been reported. In some cases, proteases do not accept a complex pharmacophore directly attached to the end of a peptide sequence. For example, in the early 1980s, vLK-doxorubicin prodrug was developed to treat cancer, but it turned out to be a poor substrate for plasmin, resulting in an unfavorable therapeutic effect, probably because of steric hindrance. To overcome this problem, self-immolative linkers were inserted between the peptide substrate and the drug molecule (37). Two types of selfimmolative systems are known: electronic cascade spacers and cyclization spacers. Paclitaxel prodrugs prepared with both spacers have been reported (38). p-Aminobenzyl oxycarbonyl (PABC) and monomethylated ethylene diamine (MED) were employed as electronic cascade and cyclization spacers, respectively (Figure 3). Both conjugated prodrugs, which have significantly decreased cytotoxicity, could be readily activated by plasmin, followed by spontaneous spacer elimination and subsequent release of free paclitaxel. Interestingly, the two systems have distinct pharmacokinetic properties. Prodrug incorporated with the PABC spacer had a slower enzymatic hydrolysis (42 min vs 3.5 min) but a faster spacer elimination rate (instantaneous vs 23 h) than with the MED spacer (Figure 3). Alternatively, a nonremovable enhancer can be covalently attached through an irreversible spacer to the drug molecule for better efficacy. The enhancer molecule to which the parent drug connects is carefully chosen to avoid significant loss of drug potency. For example, thapsigargin (TG) is an apoptotic agent with LD50 ) 30 nM in TSU-Pr1 prostate cancer cell (39). Covalent attachment of a 12-aminododecanoyl linker yielded an analogue (L12ADT) that retains the same efficacy as TG (Figure 4) and can be further conjugated with a PSA substrate (HSSKLQ). The resulting prodrug is nontoxic to normal cells, because the attached peptide substrate prevents it from entering the cells (35). The prodrug has demonstrated selective cytotoxicity in PSA-producing LNCaP cells, while remaining nontoxic to PSA-negative HCT-116 cell lines. Antibody-Drug Conjugates. Traditional anticancer drugs are highly toxic and have limited specificity to cancer cells. In
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Figure 3. Example of paclitaxel prodrug derivatives incorporated with self-immolative spacers. (A) Paclitaxel prodrug analogues were designed by incorporating PABC to paclitaxel via a 2′-carbonate. PABC is inserted between the peptide substrate (vLK) and paclitaxel. The prodrug is proteolyzed by plasmin, followed by spontaneous solvolysis. (B) The paclitaxel-2′-carbamate derivative consisting of a MED spacer and tripeptide (aFK) is hydrolyzed, followed by spontaneous cyclization reaction to yield the pentacyclic urea (38).
Figure 4. Chemical structures of (A) thapsigargin (TP), (B) 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), and (C) a PSA substrate (HSSKLQL) conjugated with 12ADT (35).
contrast, monoclonal antibodies (mAbs) can selectively bind to tumor cells via specific surface markers, but only display moderate antitumor activity. Antibody-drug conjugates (ADCs) have been designed by covalently attaching cytotoxic agents to mAbs. This tumor-targeting technology utilizes the fact that mAbs can help to deliver the nonspecific toxic agents to the tumor sites, thus greatly reduce their undesired systemic toxicity. Moreover, the mAbs can also synergic elucidate the tumor cells. To design an ADC, a linker is required to connect the cytotoxic agent and the mAb. This linker should be stable in blood circulation but readily cleaved or disconnected to release the cytotoxic drugs. Various linkers have been designed and synthesized to enhance drug releases upon cellular internalization. These include the hydrazone-based or ester linkers that can be hydrolyzed in the acidic environment of lysosomes (40-45) and the disulfide exchange or self-immolative disulfide linkers that can be reduced by glutathione inside the cells (46-48). Peptide substrates have also been employed for designing ADCs; the resulting conjugates can be hydrolyzed by lysosomal proteases such as cathepsin B to release the active drugs (45, 49, 50). For example, n-butyl diacetate derivative of doxorubicin has been synthesized and attached to both the anti-CD70 and antiCD30 mAbs via a dipeptide linker. A self-immolative PABC
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Figure 5. Schematic presentation of a prodrug-polymer. Table 2. Examples of Polymeric Drug Carriers That Are Activated by Target Proteasesa polymer carrier
active drug
peptide linker
CM-Dextran-PA Dextran HPMA HPMA L-PG PEG PGC
Exatecan (147) Methotrexate (58) Thapsigargin (148) TNP-470 (149) Paclitaxel (150) Camptothecin (56) Porphyrin (110)
GGFG PVGLIG SSKYQL GFLG EE GLFG KK
target protease indication Cathepsin B MMP-2, MMP-9 PSA Cathepsin B Cathepsin B Cathepsin B Cathepsin B
anticancer anticancer anticancer anticancer anticancer anticancer anticancer
a Abbreviations: CM-Dextran-PA, carboxymethyldextran polyalcohol; L-PG, polyglutamic acid; PEG, poly(ethylene glycol); PGC, poly(L-lysine) grafted with monomethoxy-poly(ethylene glycol) copolymer; TPN-470, O-(chloracetyl-carbamoyl) fumagillol.
spacer was further introduced in between the dipeptide linker (valine-citrulline) and the daunosamine nitrogen of the doxorubicin derivative. Upon proteolysis, 2-pyrrolinodoxorubicin was self-eliminated as the active metabolite. The ADCs were >70fold more potent than doxorubicin and also demonstrated >40fold specificity when evaluated against their corresponding antigen negative cell lines (51). Besides aiming for intracellular delivery of cytotoxic agents, peptide linkers can be employed to improve the therapeutic index of radio-immunotherapy by accelerating the liver clearance of radiometals from their immunoconjugates (ICs) (52), since cathepsin degradation of the peptide linkers could release the chelated radiometals into the circulation and subsequently eliminated by renal clearance. Cathepsin-degradable peptidelinked radio-ICs (RICs) and their corresponding 2-iminothiolane (2IT) nondegradable linked RICs have been designed to compare their pharmacokinetics in HBT-3488 human breast adenocarcinoma xenografts (53). The results indicated that the cumulative activities of peptide-linked RICs in liver were significantly lower (reduced from 59% to 68%) than their corresponding 2IT-linked RICs. Protease-Responsive Carriers and Biomatrices. Polymeric Carriers. To extend the in vivo half-life of a therapeutic agent, multiple drugs can be covalently attached to the backbone of a polymer via peptide substrates. In general, drug-peptide conjugates are covalently attached to a synthetic polymer. In the presence of a target protease, the prodrug-polymer releases multiple peptide-drug fragments that subsequently hydrolyze into active metabolites (Figure 5). Various protease substrates have been reported as the linkers (examples in Table 2). The advantages of employing polymers as drug carriers are that they can be biocompatible, biodegradable, and soluble. Polymer-drug conjugation has become a popular approach to cancer delivery, as polymers with high molecular weights tend to accumulate at tumor sites via the enhanced permeability and retention (EPR) effect, or so-called passive targeting (54, 55). For example, 10amino-7-ethylcamptothecin (SN-392) was conjugated to a linear methoxypoly(ethylene glycol) (mPEG) through a tetrapeptide (GLFG) spacer (56). The resulting polymeric conjugate had
higher antitumor activity than free SN-392 in a Meth A fibrosarcoma (solid tumor) model. Another example is dextranPVGLIG-methrotrexate conjugate. The prodrug was cleaved by both MMP-2 and MMP-9 (57), which could release a sufficient number of peptidyl methotrexate fragments to inhibit tumor growth (58). However, the tumor targeting mechanism relied predominantly on EPR. The same approach has been applied to other diseases, such as choroidal neovascularization (CNV) (59) and myocardial infarction (60), because the affected tissues also exhibit the similar anatomical characteristics to those of a tumor (i.e., hyperpermeable vessels and lack of effective lymphatic drainage). Biomatrices. Hydrogels have been extensively developed for drug delivery and tissue engineering. They are usually three-dimensional networks formed by polymeric chains cross-linking via hydrophobic, electrostatic, and/or covalent interactions; for example, naturally derived polymers such as dextrans and gelatin have been used for protein delivery (61, 62). The state of the art in tissue engineering is to optimize matrix degradation for tissue regeneration processes, while allowing healing and remodeling to take place. Some of the employed matrices are natural and can be intrinsically digested by proteases. For example, the release of vascular endothelial growth factor (VEGF) from heparinized collagen matrices appeared to be controlled by both the presence of heparin from the matrices and the proteinases that are secreted from the fibroblasts (63). To modulate (increase or decrease) the rates of matrix degradations, reagents such as glutaraldehyde (64), polyepoxy compounds (65, 66), and carbodiimides (67) have been employed to cross-link the collageneous materials. Alternatively, the amino acid side chains on the collagen could be capped to prohibit cross-linking. For example, the lysine and arginine amino acid side chains on collagen were chemically modified by n-butylglycidyl ether and methylglyoxal, respectively (68). The modified collagen had a significant reduction in swelling property, but an increased solubilization when treated with proteases such as trypsin, acetyltrypsin, and cathepsin B, which was attributed to disruption of the collagen structure by chemical modifications. Depending on the desired applications, environmentally sensitive hydrogels can be designed by chemically modifying their polymer components (69). Therapeutic molecules can be attached, encapsulated, or absorbed onto these matrices. Of particular importance, external stimuli such as temperature, electric fields, light, pH, or enzymes can be exploited to control their release rates. Various matrices that are sensitive to proteases have also been developed. An early example was the development of polyvinyl alcohol (PVA) hydrogels for bacterial prophylaxis (70). This approach was similar to that for polymeric-peptide conjugate, except an insoluble PVA matrix was grafted to gentamicin through multiple thrombin substrates (GfPRGFPAGG). Gentamicin molecules were selectively released from the matrix when incubated with thrombin-expressed Staphylococcus aureus infected wound fluid, while no biologically active antibiotic was released with noninfected wound fluid (71). Further evaluation in animals showed that the matrix was potent and specific for treating Staphylococcus aureus infected wounds (70). MMPs-activatable wafers were developed for local delivery of chemotherapeutic agent (72). Cisplatin complexed with an MMP substrate (CGLDD) was incorporated into poly(ethylene glycol) diacrylate hydrogel wafers. Drug release was dependent on MMPs expressed in a conditioned U-87 MG cell culture media. A protease-responsive hydrogel based on the KLD-12 peptide (Ac-KLDLKLDLKLDL) was reported to control release of cytotoxic peptide (73). The peptide was able to form a self-
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Figure 6. The design of a urokinase activatable hydrogel. (A) A self-assembling peptide (KLD-12) inserted with urokinase substrate was complexed with a cytotoxic peptide (kldlkl-r7-kla) to form a hydrogel system. (B) Upon addition of the targeted protease, the enzyme digests the preparations at the substrate cleavage site resulting in release of gel fragments and therapeutic peptides (77).
Figure 7. The design of a protease-cleavable LMWG-(model) drug conjugate system. Gel fiber formation is completely reversible by temperature and pH (79).
assembled matrix via β-sheet stacking (74, 75). An urokinase substrate sequence (SGRSANA) (76) was inserted between the assembling motifs with D-configuration, thus creating a uPAresponsive formulation when assembled (Figure 6). Cleavage of the protease substrate from the assembled matrix fragmented the building blocks, which in turn weakened the β-sheet interactions and caused matrix degradation. Further anchoring a cytotoxic peptide (r7-kla) into the matrix demonstrated that drug release was triggered by urokinase (77). Recently, a recombinant gelatin (HU4) containing part of the amino acid sequence of the R1-chain from human type I collagen was synthesized as the polymeric construct for hydrogel (78). The HU4 gelatin was further modified with methacrylate residues, thus enabling chemical cross-linking and gel formation by radical polymerization. The assembled hydrogels were degraded by MMP-1. In addition to polymer gel based systems, drug delivery based on low molecular weight gelator (LMWG) has been synthesized (79). A model protease-activatable prodrug, L-phenylalanyl-amidoquinoline, and two ethylene glycol chains were conjugated to a cyclohexane trisamide gelator (Figure 7). The gel fiber system demonstrated a two-stage enzyme-mediated drug release mechanism. At room temperature, the assembled gel fibers protected the prodrug molecules from protease cleavage. When the temperature was raised (from 25 to 45 °C),
the gel-to-solution transition equilibrium was shifted, and relaxed gelators became available for R-chymotrypsin cleavage to release the free 6-aminoquinoline. Protease-Sensitive Imaging Probes. Immunohistological analysis showed high regional expression of disease-associated proteases in many disease tissues. Compared with adjacent normal tissues, the difference is significant; for example, in the case of colon adenoma, upregulated cathepsin B was observed in early phase tumor (80). Labeled antibodies against enzymes are suitable for ex vivo tissue analysis, but for in vivo detection of proteases, a more sensitive sensing approach would be favored. Since the proteases are catalytic enzymes, one protease could specifically activate tens, hundreds, or even thousands of its substrates. Amplification of the signal could be expected if the molecular probes are designed to be the substrates of their target proteases (81). The ability to repeatedly and noninvasively map protease expressions in real time in vivo can be an important tool for disease diagnosis (82). Recently, three major types of activatable fluorescent probes have been developed to report protease activity in living biological systems (Figure 8) (83). To distinguish background signal from true signal, in general, the principle mechanism of activation is based on quenching-dequenching upon enzymatic cleavage. The molecular probes are typically designed to be maximally quenched
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Figure 8. Schematic diagram of protease-activatable fluorescence probes. (A) Peptide-based enzyme sensitive probe. The molecular probe has a fluorescent donor at one end of the peptide substrate and a light absorber at the other end, (B) Self-quenched enzyme sensitive probe. Fluorochromes were covalently coupled to a polymer backbone via a synthetic peptide substrate (green line). Due to the proximity of the fluorochromes, selfquenching occurs so that almost no fluorescent signal can be detected in the nonactivated state. After designated enzyme cleavage of the peptide spacer, fluorochromes are released from the carrier and become brightly fluorescent. (C) Fluorogenic enzyme-sensitive probe. Fluorescence signal will be switched on when the probe is proteolytically activated.
in their native states and to brightly fluoresce in their cleaved states. On the basis of this quenching design, the background signal can be maintained at a minimum. The first approach (Figure 8A) to producing a proteolysisactivatable probe is to flank an enzyme substrate with a fluorophore and a spectrally matched “quenching” molecule that effectively absorbs the energy from the fluorophore via fluorescence resonance energy transfer (FRET) (84). The second type (Figure 8B) of design is also based on energy transfer, but between the same types of fluorochromes. High numbers of fluorochromes were anchored onto a polymer template through cleavable peptide substrate sequences, which brought in protease selectivity. Loading the polymer with a large number of fluorochromes, typically 20-30 fluorochromes, results in nearly completely quenched fluorescence. The third type of proteasesensitive probe has the simplest design (Figure 8C). Only one fluorogenic reporter is needed for the probe. Typically, the intact probe emits no fluorescence until the amide bond between the peptide substrate and the dye is clipped. Peptide-Based Molecular Probes. Various fluorochrome and quencher pairs have been selected for the best fluorescence energy transfer (Figure 8A). By linkage through a peptide substrate sequence, molecular probes could be selectively activated. A near-infrared fluorochrome Cy5.5 pairing with an absorber NIRQ820 was developed for MMP-7 imaging. Up to a 7-fold increase in fluorescence signal was observed when treated with MMP-7 (85). A recent example with Cy5.5/BHQ-3 pair was prepared to image osteoarthritis associated MMP-13 activity and to monitor therapeutic response in living animals (86). Different from an optical quenching approach, a cell retention strategy was designed by integrating proteolysis and cell penetration signals (87). A polyarginine-based, cell-penetrating peptide was linked to a polyglutamate through an MMP-2/9 linker. Because of the intramolecular charge interaction, the cellpenetrating property was blocked until tumor-associated MMP2/9 degraded the linker and then exposed the cell-penetrating peptide, which was able to direct the optical reporter into high MMP expressing tumors, reporting the location of the tumor.
In addition to imaging, the same approach can be applied to provide site-selective drug delivery. In a separate approach, regional proteolysis was applied to change the solubility of an imaging probe, resulting in local deposition of reporter (88). An MMP-7 substrate peptide was tethered with a chelated gadolinium (Gd) and a hydrophilic PEG chain. Upon proteolytic degradation, the PEG solubility enhancer was eliminated, causing solubility to drop in the Gd-containing peptide fragment. In vivo MR imaging indicated that Gd deposition correlated with MMP-7 expression and a protease inhibition effect by therapeutic inhibitor could be conveniently imaged by MR imaging. Polymeric-Peptide Conjugate. The first protease-activatable optical contrast agent developed for in vivo imaging was reported in the late 1990s (89). The probe was a protected grafted copolymer (PGC) composed of a poly(L-lysine) backbone (PL) conjugated with multiple mPEGs and near-infrared fluorophores (Cy5.5). The fluorophores were enclosed in proximity resulting in significant quenching in their fluorescence emissions. Broad-spectrum proteases including cathepsin B and L could digest the polymeric backbone through the remaining free lysine residues, release the polymeric fragments, and subsequently initiate a dequenching process (i.e., fluorescent amplification). The approach was employed to image diseases such as cancer (89), arthritis (90), and atherosclerosis (91). To target different proteases, selective peptide substrates have been inserted between the fluorophores and the PGC (Figure 9). Simply by replacing the peptides, imaging probes were developed for detecting other disease-associated proteases such as cathepsins (92, 93), MMPs (94, 95), caspases (96), and thrombin (97). For example, a peptide substrate (GGSGRSANA) was employed for a uPA-sensitive probe (98). When the probe was tested in HT-1080 breast fibrosarcoma tumor-bearing mice, the observed fluorescence signals at the tumor sites were correlated with uPA activity (99). Recently, self-assembled nanofibers have been designed to sense uPA activities (100). Their basic assembly constructs comprise (1) a peptide sequence (kldlkldlkldl) reported to form stable β-sheet platform; (2) a uPA substrate motifs (SGRSANA); (3) a nontoxic
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Figure 9. Schematic presentation of a urokinase-selective probe. The Cy5.5-peptide substrates (Cy5.5-GGSGRSANAKC) are attached to a PGC. The close spatial proximity of these multiple conjugates leads to fluorescence quenching. Upon urokinase cleavage, the released peptide fragments result in fluorescence signal amplification (98).
MPEG attached at the peptide N-terminus; and (4) a fluorescein as the optical reporter. Upon addition of proteases, the enzymes digest the nanofibers via their substrate cleavage sites, causing the release of hydrophilic fluorescent fragments and resulting in fluorescence emission. Interestingly, these nanofibers have the ability to compensate for the loss of intrinsic hydrophilicity; the remaining digested or partially cleaved nanofibers undergo morphological changes with time (101). Fluorogenic Probes. On the basis of delocalization of the lone pair electrons, fluorogenic probes can be extremely simple (Figure 8C). Amino-4-methylcoumarin (AMC) is a widely used fluorogenic dye in measurements of protease activity. For in vivo applications, fluorophores having the excitation and emission at near-infrared (NIR) region (700-1000 nm) are generally preferred, since they have less interference from endogenous chromophores and also have deeper tissue penetrations while imaging. A series of fluorogenic dyes with benzo[a]phenoxazine core structure have been synthesized and evaluated (102, 103). Among them, 9-di-3-sulfonyl-propylaminobenzo[a]phenoxazonium perchlorate (2SBPO) has the best solubility and optical properties. Thus, it was selected as a scaffold to synthesize a panel of enzyme substrates, sensitive to amino peptidase (103), other proteases (104), and galactosidases (105). The assembled probes are nonfluorescent but display strong fluorescent emission at 670 nm following proteolytic release of 2SBPO. Bioluminogenic probe is another type of optical imaging agent designed by using the same concept as fluorogenic substrate. Peptide substrate was added to amino-luciferin, which was derived from the natural substrate of fire fly luciferase, D-luciferin. Because of steric hindrance, luciferase cannot recognize the peptidemodified luciferin. After proteolytic hydrolysis of the peptide, free luciferin was released and utilized by luciferase in situ. We have used a commercially available DEVD-luciferin substrate to study treatment effect of anticancer gene therapy (106). Expressed anticancer protein induced apoptosis of tested tumor cells, triggering caspase-3 expression. A bioluminescence signal was found associated with the gene therapy effect.
Protease-Mediated Photodynamic therapy. Photodynamic therapy (PDT) is currently used to treat several types of cancer. Regional therapeutic effect can be achieved only by combining photosensitizer (PS), light, and oxygen. Each component is harmless by itself, but when all three components get together, light excitation of a PS will locally generate highly cytotoxic singlet oxygen, 1O2, which induces cell death and tumor ablation (107). Most PSs are porphyrin analogues that absorb light energy and then transfer to oxygen or emitted fluorescence. It has been reported that singlet oxygen generation is closely correlated with fluorescence intensity (108). When a PS is in a fluorescent quenched state, its ability to generate singlet oxygen is also reduced. Using the same PGC platform used in imaging probes, a proPDT agent was synthesized by replacing Cy5.5 with a photosensitive agent, chlorin e6 (Ce6). When the assembled agent was in its native state, both the fluorescence and the singlet oxygen generation (SOG) of the anchored Ce6 were depopulated by energy transfer (Figure 10). The resulting pro-PDT agent had a lower toxicity (13%) than the free Ce6, but the hidden phototoxicity could be recovered after proteolysis (109). In an animal experiment with one single low dose of pro-PDT agent, tumor growth was slowed by 50% (110). In addition, local Ce6 concentrations could be simultaneously monitored by fluorescence imaging. A similar polymer-induced quenching approach was also reported, using pheophorbide-a as the PS (111). Phototoxicity was recovered when treated with a model protease, trypsin. Using the same concept as previously described for peptide-based molecular probes, short peptide-based proteasemediated PDT agents were also developed with a pyropheophorbide-a photosensitizer and a BHQ-3 quencher pair (112). Protease-mediated phototoxicity of target enzymes, caspase-3 and MMP-7, was demonstrated in vitro and in vivo (112, 113). Protease and Nanoparticles. Particle sizes under 100 nm are unlikely to be eliminated by renal filtration but rather cleared by the liver and spleen or phagocytized by macrophages (114). Similar to polymeric carriers, nanoparticles ranging in size from
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Figure 10. Schematic presentation of a protease-sensitive photodynamic therapeutic agent. Multiple photosensitizers (Ce6s) are attached to a PGC. Intramolecular energy transfer between the closely positioned Ce6 molecules contributes to a significant quenching of fluorescence emission and singlet oxygen generation. Following enzymatic cleavage of the poly(L-lysine) backbone, the released Ce6s-polymeric fragments regain their fluorescent properties and SOG (110).
20 to 150 nm tend to accumulate in tumor tissues via EPR effect (54). Nanomaterials such as nanofibers (115), carbon nanotubes (116), superparamagnetic iron oxide crystals (117), quantum dots (118), dendrimers (119), polymeric micelles (32), liposomes (120), nanospheres (121), and polyplexes (122) have been engineered as therapeutic carriers. Efficient drug delivery can be achieved by incorporating therapeutic agents into these inherent carriers (55). Some of them are designed to be degraded by natural proteases. For example, paclitaxel-loaded gelatin nanoparticles were developed for intravesical therapy of superficial bladder cancer (123). The particles are rapidly degraded by Pronase to release the free drug molecules. To promote cellular internalization, protease-activatable liposomes were designed by conjugating an acetylated dipeptide substrate (N-Ac-AA) through its carboxyl terminus to the amino group of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (124). The resulting peptide-lipid (N-Ac-AA-DOPE), together with dioleoyl trimethylammonium propane (DOTAP) and phosphatidyl-ethanolamine (PE), were assembled into nonfusogenic liposomes. The liposomes were activated by elastase or proteinase K to become fusogenic for enhanced intracellular delivery (Figure 11). In another study, a pegylated MMP-2 substrate (GPLGIAGQ) was conjugated to the amino group of DOPE (125). When assembled with disteraoyl phosphatidylcholine (DSPC), cholesteryl chloroformate, and cholesten-5-yloxy-N-(4-((1-imino2-,β-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4Chol) (126), the resulting liposomes (Gal-PEG-PD) were composed of (1) active targeting ligands (galactose moieties) recognized by the hepatic cell surface asialoglycoprotein receptors; and (2) a pegylated peptide substrate cleaved by MMP-2 (Figure 12). In the absence of enzymes, Gal-PEG-PD-liposomes could not be taken up by normal hepatocytes because of steric shielding of the targeting ligands. With the addition of MMP2, the enzyme removed the bulky mPEGs, thus exposing the galactose ligands for targeting. The cytotoxicity of N (4)-
Figure 11. (A) Chemical structure of N-Ac-AA-DOPE. The lipid-peptide substrate can be converted into DOPE by elastase (124). (B) Activation of a non-fusogenic liposome. The liposome is stable with negatively charges. Removal of N-Ac-AA by elastase results in charge reversal, thus enhancing the ability to fuse with the plasma membrane for drug delivery.
octadecyl-1-β-D-arabinofuranosylcytosine (NOAC) incorporated Gal-C4-PD liposomes were significantly enhanced in cultured HepG2 cells after pretreatment with purified human MMP-2. Quantum dots (QDs) that are activated by MMP-2 and MMP-7 were developed for cellular imaging (127). The QDs were attached with a peptide consisting of (1) a cationic tetraarginine as the transporter for intracellular delivery; (2) an anionic tetraglutamic acid as the blocker to inhibit cellular uptake; and (3) a peptide substrate sequence (PLVGR). Once the blockers are removed by MMPs, resurfaced tetraarginine signals direct QDs into cells. FRET pairs composed of QDs are also designed to sense protease activities (128, 129). Quenchers such as gold nanoparticles (128), organic fluoro-
Reviews
Figure 12. Schematic presentation of MMP-2 activatable targeting liposome (126). Cell-specific delivery was initiated when the masking PEG chain was eliminated by MMP-2.
phores (129), or fluorescent proteins (130) have been attached to the QDs via peptide substrates. QD photoluminescence was quenched through FRET. Protease cleavage of the peptide linkers released the quenchers, which subsequently recovered the QD luminescence emissions. This approach has been applied to study the protease activity in HTB126 cancer cell line (131). A low nanometer-sized dendritic molecular probe having Cy5.5 as fluorescence reporter and AF750 as an internal reference was prepared for tumor detection (132). The AF750 was attached to a dendrimer directly, while Cy5.5 was linked through an MMP-7 spacer. In animal experiments, a bright Cy5.5 signal switched on by intestinal adenoma-associated MMP-7 was found in microsized adenomas. In another dendrimer example, anticancer agent Pt2+-complex was attached to first-generation dendrimers through a cathepsin B releasable linker (133). Cell culture experiments confirmed the potential of applying this dendritic drug in anticancer therapy. Organic nanoparticles have been formed by a “hydrophobic collapse-like” construct (134). In aqueous solution, a hydrophobic core was formed by multiple deoxycholic acids and was attached to a branched polyethyleneimine (PEI) chain. Multiple fluorescence-labeled caspase-3 substrates were then tethered to the surface of spherical particles, forming 80-100 nm organic nanoparticles. Because of a self-quenching mechanism, the fluorescence signal was shielded until the particle was recognized by target proteases. The described nanoprobe has been applied to following the apoptotic process under a fluorescence microscope.
CONCLUSION AND PROSPECTS Proteolysis is a simple reaction that cleaves the peptide amide bond, but its applications have been proved enormous. Taking advantage of disease associated proteolysis, numerous prodrugs, biomaterials, and probes have been developed. Pharmacophores and reporters are first restrained or masked by various smart designs. Subsequent proteolysis by target proteases releases the active ingredients chemically or physically, regaining the desired therapeutic activity or imaging sensitivity in situ. Another advantage of proteolysis in drug development is catalytic capability. Operating like a self-propelled machine, one protease could potentially release a large number of drugs. Moreover, the catalytic function is a kind of in vivo PCR reaction that amplifies signal outputs and improves the detection limit significantly, which may have tremendous potential medical applications including disease detection and diagnosis.
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In this brief review, only proteolysis was summarized, but it is not the only enzyme candidate used in drug engineering. Other natural enzymes have also been proposed, studied, and applied. For example, lactamase-responsive prodrugs (135), hydrogel biomaterials (136), and imaging probes (137) have all been reported. Another good example is transglutaminase, a type of enzyme usually found at the site of tissue healing and blood coagulation. It cross-links proteins through glutamate and lysine side chains. On the basis of this unique chemical linking property, transglutaminases are often used to increase toughness of hydrogels that can be applied to drug delivery systems or implants for tissue regeneration (138, 139). Imaging probes deposited by transglutaminase were also developed to understand healing and blood clotting processes (140-142). Many more enzymes have been identified but are waiting for creative twists, directing their enzymatic capability to assist in diagnosis and therapy. Since disease-associated enzymatic activities vary during disease progression and between individuals, an enzymeresponsive delivery system potentially could provide an in situ self-regulated medicine, paving the way to personalized medicine. It can be envisioned that more and more intelligently designed enzyme-responsive systems will be developed for their environmental sensitivity. To achieve the ultimate goal, treating individual patients with personalized medicine, a more profound understanding of enzyme structure, mechanisms, distribution, and regulation in vivo is required.
ACKNOWLEDGMENT This research was supported in part by RO1-CA099385 and RO1-CA135312 to CT, and North Dakota EPSCoR Program (FAR0014970) to B.L.
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