Enhanced Prostate Cancer Targeting by Modified Protease Sensitive

May 1, 2012 - Prostate cancer (PCa) is the most frequent cancer in men and the second most frequent cause of cancer related death in men.(1) In the pa...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Enhanced Prostate Cancer Targeting by Modified Protease Sensitive Photosensitizer Prodrugs Maria-Fernanda Zuluaga, Doris Gabriel,† and Norbert Lange* Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland S Supporting Information *

ABSTRACT: Prodrugs combining macromolecular delivery systems with site-selective drug release represent a powerful strategy to increase selectivity of anticancer agents. We have adapted this strategy to develop new polymeric photosensitizer prodrugs (PPP) sensitive to urokinase-like plasminogen activator (uPA). In these compounds (to be referred to as uPA-PPPs) multiple copies of pheophorbide a are attached to a polymeric carrier via peptide linkers that can be cleaved by uPA, a protease overexpressed in prostate cancer (PCa). uPA-PPPs are non-phototoxic in their native state but become fluorescent and produce singlet oxygen after uPA-mediated activation. In the present work, we studied the influence of side-chain modifications, molecular weight, and overall charge on the photoactivity and pharmacokinetics of uPA-PPPs. An in vitro promising candidate with convertible phototoxicity was then further investigated in vivo. Systemic administration resulted in a selective accumulation and activation of the prodrug in luciferase transfected PC-3 xenografts, resulting in a 4-fold increase in fluorescence emission over time. Irradiation of fluorescent tumors induced immediate tumor cell eradication as shown by whole animal bioluminescence imaging. PDT with uPA-PPP could therefore provide a more selective treatment of localized PCa and reduce side effects associated with current radical treatments. KEYWORDS: polymeric prodrugs, protease-sensitive prodrugs, photodynamic therapy, urokinase-like plasminogen activator, prostate cancer

■

1990,9 an increasing number of studies have been published.10−19 Besides light penetration depth and tissue oxygenation, a current limitation of PDT for PCa is the heterogeneity of response (presumably related to heterogeneity of PS uptake itself).6 Moreover, prolonged skin sensitivity9,10 and extraprostatic treatment effects17,18 have been reported as major disadvantages in clinical PDT of PCa. Among the strategies to improve biodistribution and tumor selectivity of PS, one approach exploits characteristic changes in the vasculature of solid tumors to improve drug delivery via the enhanced permeability and retention (EPR) effect.20 Besides altered vascular architecture, many tumors are shown to have elevated levels of proteases presumably in adaptation to rapid cell cycling; repression of important regulatory proteins; and sustained invasion, metastasis, and angiogenesis.21 Because these proteolytic enzymes are present at high levels in tumors, they represent an attractive target for tumor imaging and prodrug activation. We and others have translated these concepts into PDT by the development of macromolecular protease-sensitive photosensitizer prodrugs (PPPs).22−24 In these prodrugs intramolecular interactions between closely positioned PS units efficiently inactivate the photoactivity in the

INTRODUCTION Prostate cancer (PCa) is the most frequent cancer in men and the second most frequent cause of cancer related death in men.1 In the past decades, intensive research in this field has led to remarkable changes in diagnosis and treatment.2 Due to widespread PCa screening, most patients are diagnosed at the stage of early, localized disease.2−4 Therefore early radical treatments can be offered to the majority of patients. Current treatments for localized PCa, such as radical prostatectomy and external beam radiation therapy, show a strong survival benefit for men with high risk disease.5 However, these radical treatments are associated with substantial morbidity and decreased quality of life.6 Patients' continence (urinary and intestinal) and sexual function are particularly affected. In principle, these adverse effects might be reduced by increasing the selectivity of the treatment and by avoiding damage to structures that surround the prostate (including the rhabdosphincter, neurovascular bundles, rectum and ejaculatory apparatus). Photodynamic therapy (PDT) has the potential to fill some of the gaps in current treatments.7 PDT combines the simultaneous use of a photosensitizer (PS), oxygen and light to locally generate cytotoxic reactive oxygen species (ROS) eradicating unwanted tissues. PDT was first used clinically for superficial conditions; nevertheless, nowadays, the combination of lasers with appropriate light delivery systems makes it possible to apply it to hollow and parenchymatous organs.8 Since the first report of PDT treatment of PCa in © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1570

November 14, 2011 April 27, 2012 May 1, 2012 May 1, 2012 dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

Fisher Scientific (Erembodegem, Belgium). D-Luciferin Firefly, potassium salt was purchased from Biosynth AG (Staad, Switzerland). Synthesis. PPPs were synthesized in three steps as described in detail elsewhere.29 To obtain a uPA-sensitive conjugate, the L-configured peptide GSGRSAG containing the reported urokinase minimal substrate27 and a corresponding Dconfigured control peptide were synthesized using standard Fmoc chemistry. Subsequently, NHS-activated Pba was coupled to the N-terminus of the peptides and the corresponding Pba−peptides conjugates were purified by preparative RP-HPLC (Waters Delta 600 HPLC) on a C8, Nucleosil 300-10 column (Macherey−Nagel) using a 0.1% TFA/water/acetonitrile gradient, and molecular mass was analyzed by ESI-MS, with a Finnigan MAT SSQ 7000 (Thermo Electron Co. Waltham, MA). Final uPA-PPPs were synthesized with a previously optimized Pba−peptide loading of 25 units per 100 free epsilon-NH2 groups of the PL. For this purpose, Pba−peptide (3.06 mg, 3.1 × 10−6 mol), PL 18 kDa (2.00 mg, 0.11 × 10−6 mol, 1.1 × 10−5 mol of −NH2 functions), and HATU (1.36 mg, 4.03 × 10−6 mol, 1.3 equiv based on Pba−peptides to be activated) were dissolved in DMSO (0.65 mL), and DIPEA (3.7 mg, 3.3 × 10−5 mol, 3 equiv of free −NH2 functions of PL) was added to the stirred solution. The reaction was carried out in the dark under argon for 4 h at room temperature. Complete loading of the Pba−peptides on PL was confirmed by analytical RP-HPLC as described in ref 29. The polymeric carrier was further modified to obtain 5 different uPA-PPPs (see Table 1). Modifications were achieved

prodrug state. The delivery vehicle should passively guide high payloads of inactive PS to the target site via the EPR effect. Activation of prodrug is then trigged in response to proteasemediated release of PS moieties and leads to the restoration of its fluorescent and photodynamic activity. Urokinase-like plasminogen activator (uPA) is a serine protease believed to play a key role in tumor progression in PCa.25,26 Plasma levels of uPA and its receptor (uPAR) have been found to be significantly higher in patients with clinically localized (0.34 ng/mL; 1.6 ng/mL, respectively) and metastatic PCa (0.40−0.60 ng/mL; 1.9−2.4 ng/mL, respectively) than in healthy men (0.20 ng/mL; 1.5 ng/mL, respectively). These higher circulating levels of uPA and uPAR probably are at least in part prostatic in origin because they decrease significantly after prostate removal (0.24 ng/mL; 1.5 ng/mL, respectively).26 Furthermore, immunohistochemical staining of prostate tissue microarray sections has shown overexpression of both proteins uPA and uPAR in primary cancer specimens and metastasis in a large set of human PCa samples but not in normal and benign prostate tissues.25 uPA-PPPs have dual functionality, to make lesions fluorescent in response to uPA activity and to exert a selective cytotoxic effect upon irradiation with light. Our lab has previously evaluated an established uPA-peptide substrate27 for the development of a uPA sensitive prodrug and characterized the influence of charge of the polymeric backbone on in vitro activation.28 To advance the application of these prodrugs to in vivo tumor imaging and therapy, in the present study we have redesigned the conjugates to achieve tailored pharmacokinetic properties. Synthesis, characterization, and activation of different uPA-PPPs based on a poly-L-lysine (PL) backbone with different side chain modifications (mPEG 20 kDa, mPEO4 and mPEO8) are described. Fluorescence imaging allowed visualization of tumor homing properties and enzymatic activation of prodrugs in a PCa xenograft model. Furthermore, preliminary PDT studies in the same model are also reported.

Table 1. Calculated Composition (Average Units per 100 Lys ε-Residues) and Estimated Mean Molecular Weight of uPA-PPPs

■

EXPERIMENTAL SECTION Chemicals. Anhydrous forms of dichloromethane (DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (ACN), diethyl ether and trifluoroacetic acid (TFA) were purchased from Acros Organics (Geel, Belgium). HGly-2-chlorotrityl resin (1.1 mmol/g), Boc-glycine, Fmocglycine, Fmoc-alanine, triphenylisopropylsilane, Boc-D-glycine, Fmoc-D-alanin, N,N-diisopropylethylamine (DIPEA), piperidine, picrylsulfonic acid aqueous solution (1 M), sodium iodide and ethanol were obtained from Fluka (Buchs, Switzerland). The D- and L-amino acids Fmoc(tBu)-serine, Fmoc(Pbf)arginine, as well as O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) were purchased from Genscript (Piscataway, USA). Poly-L-lysine HBr (PL; 18 kDa and 45 kDa), poly-D-lysine (20 kDa), and RMPI 1640 growth medium were provided by Sigma-Aldrich (Buchs, Switzerland). Urokinase (high molecular weight, human urine) was obtained from Calbiochem/VWR (Zug, Switzerland). Pheophorbide a (Pba) was purchased from Frontier Scientific (Carnforth, U.K.). mPEG-SPA (20 kDa) was purchased from Nektar (San Carlos, USA). HBSS, D-PBS, and TrypLE Express were purchased from Invitrogen (Basel, Switzerland). mPEO4NHS and mPEO8-NHS, as well as MEM/EBSS, sodium pyruvate 100 mM solution, MEM vitamin solution, L-glutamine 200 mM solution, MEM nonessential amino acids solution and defined fetal bovine serum (FBS), were provided by Thermo-

uPAPPP

Pba− peptide

mPEG20

1 2 3 4a 5b

25 25 25 25 25

1 2 1 1 2

NICO mPEO4 mPEO8 74 73 74 74 73

∼MW (kDa) 80 100 80 90 220

a

A homologous D-control conjugate was also synthesized. b45 kDa PL was used instead of 18 kDa PL.

by the covalent coupling with high molecular weight mPEG chains and, second, by capping the remaining epsilon-lysine residues with the following moieties: (1-methyl-3-pyridinio) formate iodide (NICO); methyl tetraethylene oxide (mPEO4); or methyl octaethylene oxide (mPEO8). For this propose, mPEG-SPA 20 kDa (1.91/3.83 mg, 9.56/19.1 × 10−8 mol, 1.1 equiv based on the number of −NH2 of PL in 0.2 mL of DMSO) was added to the PL-Pba−peptide solution under stirring and at 19 °C. The reaction was kept in the dark and left to proceed overnight at room temperature. Final modifications were achieved by adding either N-succinimidyl (1-methyl-3pyridinio)formate iodide (2.56 mg, 7.01 × 10−6 mol, 1.1 equiv of the remaining −NH2-lysine residues; 0.1 mL in DMSO), mPEO4-NHS (2.36 mg, 7.01 × 10−6 mol; 0.1 mL in DMSO) or mPEO8-NHS (3.61 mg, 7.01 × 10−6 mol; 0.1 mL in DMSO) to reaction mixtures, respectively. Reactions were stirred in the dark for 24 h at room temperature. Crude products were purified by size exclusion chromatography using a Sephacryl S1571

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

GSGR was analyzed by ESI-MS with a Finnigan MAT SSQ 7000 (Thermo Electron Co. Waltham, MA, USA). Cell Culture and Activation in Vitro. The luciferase transfected PC-3M-luc-C6 cell line of PCa origin was a generous gift from Caliper Life Sciences (Hopkinton, MA, USA). Cells were cultured in MEM/EMBSS supplemented with 10% fetal bovine serum, nonessential amino acids, Lglutamine, sodium pyruvate and MEM vitamin solution, and maintained at 37 °C in a humidified incubator containing 5% CO2. LNCaP cell line (ATCC, Manassas, VA) was cultured in RMPI 1640 growth medium supplemented with 10% FBS. Both cell lines were maintained as a monolayer and, for experiments, were harvested using TrypLE Express and resuspended in fresh complete medium. Cellular activation of prodrugs on PC-3M-luc-C6 and LNCaP was evaluated as follows: 100 μL of a cell suspension (1.2 × 105 cells/mL and 3.0 × 105 cells/mL, respectively) was seeded into 96 well plates and allowed to attach overnight. The next day, the cells were rinsed with 200 μL of HBSS and incubated with solutions of the corresponding uPA-PPP (1 μM Pba equiv) in a HBSS solution containing 10% FBS. Immediately after incubation start, the fluorescence was measured with a Safire microplate reader (Tecan, Switzerland) [excitation:emission = 400:675 nm]. Further measurements were performed 3, 6, 22−24 and 48 h later, without removal of the incubation medium. The increase in fluorescence emission was calculated by subtraction of the fluorescence intensity immediately after incubation start F0, from the value Fx obtained at time x and expressed as the ratio with respect to F0. Results were obtained in sextuplicate and expressed as mean ± SD. In Vivo Fluorescence and Bioluminescence Imaging. Female Swiss Nu/Nu mice (5−6 weeks, 17−22 g) were supplied by Charles River Laboratories (L’arbresle, France). The mice were maintained with ad libitum access to sterile food and acidified water in a light cycled room acclimatized at 22 ± 2 °C and under specific pathogen free status. All experimental procedures on animals were performed in compliance with the Swiss Federal Law on the Protection of the Animals, according to a protocol approved by the local veterinary authorities. To induce xenografts, 1.5 × 106 cells were injected subcutaneously into the dorsal region of mice. Tumors of 100−150 mm3 in size were formed within 3 weeks after inoculation. An IVIS 200 small-animal imaging system (Caliper Life Sciences Inc., Hopkinton, MA, USA) was used to quantify the PS-fluorescence in tumors. All fluorescent images were acquired with a cooled CCD camera system, using a Cy5.5 filter set (excitation, 615−665 nm; emission, 695−770 nm), a field of view (FOV) of 12.8 cm, an exposure time of 10 s, and a lens aperture of f/2. Data were analyzed with Living Image 3.0 software (Caliper Life Sciences Inc., Hopkinton, MA, USA), and the fluorescence intensity of regions of interests (ROIs) was expressed as fluorescence efficiency (emitted photons normalized to the incident excitation intensity per cm2). Prior to prodrug administration, “prescan” images were acquired for each animal to record tumor autofluorescence. This “background” fluorescence was subsequently subtracted from all further images. Mice (n = 3/prodrug) were injected systemically (retro-orbital injection) with prodrugs (100 μL, 2.0 mg/kg Pba equiv in 5% ethanol, 65% deionized water and 30% PEG 400) and imaged 3, 6, 12, and 24 h after injection under 1−2% isoflurane inhalation. Bioluminescence in vivo imaging was carried out in order to colocalize the bioluminescence produced

100 (Amersham Biosciences, Otelfingen, Switzerland) column and a mixture of acetonitrile/water/TFA (30:70:0.00025) as eluent. The fractions containing the product were pooled, lyophilized and stored light-protected at −20 °C until use. The corresponding control conjugates containing D-amino acids were obtained using the same synthetic procedure with poly-Dlysine as backbone and mPEO8 as surface modifying groups. Prodrug 5 was synthesized with a 45 kDa PL as polymeric backbone. Purity of prodrugs was confirmed by RP-HPLC monitoring at 280, 330, and 450 nm. Mass analysis of prodrug 4 and D-control conjugate 4* was performed by SEC-MALLSRI-UV using a column Waters Ultrahydrogel linear (column temperature, 35 ± 0.2 °C; mobile phase, 0.15 M acetic acid, 0.1 M sodium acetate, 0.05% NaN3 at a pH of 4.0; flux, 0.4 mL/ min).The system used contains a pump Waters Alliance HPLC system (Milford, MA), a Schambeck RI detector (Bad Honnef, Germany), a light-scattering detector Wyatt MiniDawn (Dernbach, Germany), and a UV−vis detector Waters Lambda-Max (Milford, MA). Fluorescence and ROS Quenching. Fluorescence and ROS quenching factors were determined for all prodrugs as follows: the fluorescence intensity of equimolar solutions of prodrugs (3 μM Pba equivalents) was measured at 37 °C using a SPEX Fluoromax [excitation:emission = 400:670 nm] (Perkin-Elmer, Wellesley, MA, USA). The fluorescence quenching factor (x-fold decrease of background subtracted fluorescence at the 670 nm emission maximum) was calculated with respect to the nonquenched reference conjugate loaded with 1% Pba. The relative photoinduced ROS production was indirectly determined by measuring the oxidation of I− to I3− (286 nm absorbance band) in aqueous solution.29 To 0.6 mL of D-PBS buffered solutions of uPA-PPPs (3 μM Pba equiv), 0.2 mL of an aqueous NaI solution (2.5 M) was added and the UV/Vis spectrum was recorded before and after irradiation (3.7 J/cm2). The ROS quenching factor (x-fold decrease in optical density at 286 nm) was calculated with respect to the nonquenched reference conjugate. All measurements were performed with a Cintra 40 UV/Vis spectrometer (GBC, Dandenong, Australia). Results were obtained in triplicate and sextuplicate, respectively, and expressed as mean ± SD. Enzymatic Activation. To test the enzymatic activation of the prodrug by urokinase, D-PBS buffered solutions of uPAPPPs (3 μM Pba equiv) were incubated with the enzyme (100 U) at 37 °C in the dark. Aliquots of the digestion mixture were sampled in DMSO (75%) after 5, 10, 20, 30, 60, and 120 min, and fluorescence was measured with a FluoroMax spectrofluorimeter as described above. Further control experiments were performed under identical conditions with the noncleavable D-conjugates. Fold fluorescence increase at time t is expressed as the ratio between the fluorescence emission increase after t minutes of digestion (Ft − F0) and the fluorescence emission before enzymatic digestion (F0). All measurements were performed in sextuplicate and are expressed as mean values ± SD. Moreover, the release of the Pbapeptidyl fragment Pba-GSGR after urokinase digestion (100 U, 60 min, 37 °C, in the dark) was monitored by analytical RPHPLC. Separation was performed on a Nucleodur C18 gravity 3 μm CC 125/4 column (Macherey−Nagel) using a gradient method (water/acetonitrile/TFA from 50/50/0.1 to 1/99/0.1 within 13 min). Fluorescence was detected on a Merck Hitachi FL detector L7480 [exitation:emission = 400:670 nm] (Tokyo, Japan). The molecular weight of the cleaved fragment Pba1572

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

Figure 1. Schematic structure of uPA-PPPs. The loading of Pba−peptide per polymer chain is 25% for all prodrugs (w = 25% of lysines per polymer chain). Nonfunctionalized ε-residues are modified with different moieties (x = 73%, y = 1% and z = 1% of lysines per polymer chain).

dependent fluorescence increase after systemic (retro-orbital) injection of prodrugs.

by transfected tumor cells with the fluorescence signal. Ten to fifteen minutes prior to in vivo imaging, at 2 and 24 h after injection of prodrugs, animals received the substrate D-luciferin at a dose of 150 mg/kg in DPBS by intraperitoneal injection, and bioluminescent images were obtained together with the fluorescent images using the same camera with an exposure time of 2 s. PDT on PCa Xenografts. Preliminary PDT studies were performed in PC-3M-luc-C6 xenograft bearing mice (N = 3). The animals were injected retro-orbitally with prodrug 4 at three different doses corresponding to 2.5, 5.0, and 10.0 mg/kg Pba equiv, respectively. Sixteen hours after prodrug administration, tumors were irradiated with a light dose of 100 J/cm2 at 665 ± 5 nm (Ceralas I 670, Biolitec; Jena, Germany) while the animals were maintained under 1−2% isoflurane inhalation. PDT effects were followed for 5 days by bioluminescence imaging of animals as mentioned elsewhere. Statistical Analysis. Means ± SD were used for the expression of data. Statistical analyses of data were done using Student’s t test. Nonlinear regression (curve fit Ymax and K) comparison was used to analyze fluorescence profiles after enzymatic cleavage or cellular activation of prodrugs. One-way ANOVA and Tukey analysis were used to compare time-

■

RESULTS Synthesis. uPA-sensitive polymeric photosensitizer prodrugs were synthesized in three steps: A minimal peptide substrate of uPA and a control peptide, consisting of the corresponding D-amino acids, were synthesized by conventional FMOC-solid phase peptide synthesis. ESI-MS analysis after preparative RP-HPLC of the crudes confirmed the identity of the two peptides (591.6 [M + H]+ = 591.3 calculated for C21H39N10O10+). Chemoselective coupling of Pba to the Nterminus of the peptides yielded the corresponding Pba− peptide conjugates. Their molecular mass was confirmed by ESMS analysis (1165.8 ([M + H] + = 1165.6 calculated for C57H77N14O13+) and 583.6 ([M + 2H] 2+ = 1166.6 calculated for C57H78N14O13+). Subsequently, uPA-PPPs with different backbone substitutions were assembled in a one-pot reaction. First, the photosensitizer-peptides were attached to the PL backbone at a loading ratio of 25%. Then, the nonfunctionalized ε-residues were subsequently modified. Purified products after SEC were characterized by HPLC and SECMALLS-RI-UV analysis (Figures S1 and S2 in the Supporting Information, respectively). The structures of all uPA-PPPs are 1573

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

of this compound where strong interactions between PS occur. We have already observed that although small molecular substituents of different charge do not significantly interfere with PS interactions, high degrees of substitution with polyethylene glycol groups strongly increase ROS production. This is coherent with lower ROS quenching factors calculated for the neutral prodrugs (3 and 4) and the two positively charged prodrugs (2 and 5), all with increased pegylation degree compared to prodrug 1. Enzymatic Activation. Incubation of prodrug 4 with uPA resulted in an increase in fluorescence over time and reached a plateau within 2 h (Figure 2A). As expected, this increase is significantly higher compared to the nonclevable control conjugate. The analytical HPLC traces shown in Figure 2B demonstrate that uPA-induced fluorescence increase of the prodrug is due to cleavage of the peptide linker and release of the Pba-peptidyl fragment. After cleavage of the Pba−peptide (a, retention time (tR) 4 min), a peak of the more lipophilic Pba-peptidyl fragment appears at a tR of 5 min (b). If the intact prodrug is injected, no fluorescence is visible (c). After incubation with the enzyme, a peak with a tR of 5 min appears (d). Furthermore, the corresponding D-control was not activated by uPA (e). Mass analysis of the fragment PbaGSGR (950.7, [M + H] + = 950.1 calculated for C48H61N12O9+) confirmed cleavage at the expected site between the arginine and the serine residue. Although similar fluorescence increase profiles were observed for all prodrugs after incubation with uPA (Figure S3 in the Supporting Information), significantly different maxima were reached (P = 0.0001). The positively charged prodrugs 1 and 2 showed an overall fluorescence change of 22 and 14 times, respectively. The fluorescence increase found for these two pegylated positively charged prodrugs also significantly exceeds that of the neutral compounds 3 (4 times) and 4 (3 times) as well as that of the 45 kDa PL prodrug 5 (3 times). Activation in Vitro. PPP activation by proteases expressed by PC-3M-luc-C6 was confirmed for all prodrugs by a timedependent fluorescence increase after incubation with the respective 1 μM solutions (Figure 3A). All profiles are statistically different (P < 0.0001). In contrast to incubation of recombinant uPA, the high molecular prodrug 5 showed the fluorescence increase, reaching 16 times after 46 h presumably due to the low initial background fluorescence (see Table S1 in the Supporting

graphically depicted in Figure 1. All prodrugs were designed to have a fully protected backbone (R1 + R2 = 75%), to prevent nonspecific enzymatic degradation, and were pegylated with 20 kDa mPEG to increase water solubility and to prevent a potential rapid clearance from bloodstream. Prodrugs 1, 3 and 4 have similar molecular weights [approximately 80−90 kDa], but are either noncharged (3 and 4) or positively charged (1) at physiological pH. Prodrugs 2 and 5 were synthesized in order to investigate the influence of the molecular weight increase by increasing the 20 kDa mPEGsubstitution and/or the use of a higher molecular weight PL as polymeric backbone (45 kDa PL). Estimated molecular weights are in both cases significantly higher (approximately 100 and 220 kDa, respectively). Fluorescence and ROS Quenching. Data for quenching of fluorescence emission and ROS generation of fully assembled prodrugs is summarized in Table 2. Table 2. Quenching Factors of uPA-PPPs Calculated with Respect to the Nonquenched Pba ± SD quenching factor uPA-PPP 1 2 3 4 5

fluorescence 83 84 48 61 172

± ± ± ± ±

6 3 2 3 6

ROS 15.4 9.0 10.5 8.6 10.3

± ± ± ± ±

0.6 0.2 0.2 0.2 0.3

All prodrugs showed fluorescence quenching factors in the same order of magnitude, except prodrug 5, with a 45 kDa PL instead of the 18 kDa PL as backbone, which was two times higher. This corroborates our previous studies on a homologous series of uPA-PPPs with increasing PL size chain, showing a significant decrease in fluorescence emission with increasing molecular weight (Table S1 in Supporting Information). Although these previous studies also showed decreasing ROS production with increasing backbone size, here prodrug 5 gave a similar value compared to the other prodrugs. This implies that the reduction in ROS generation could not be simply attributed to molecular size, since prodrug 1 with low molecular weight was most efficiently quenched with respect to ROS production presumably due to a particular configuration

Figure 2. (A) Fluorescence-time-profile after enzymatic cleavage of prodrug 4 (◆) and the D-control 4* (◇) by urokinase. All measurements were performed in sextuplicate and are expressed as mean values ± SD. (B) (a) Analytical HPLC trace of Pba-GSGRSAG (tR: 4 min). (b) After partial enzymatic digestion of Pba-GSGRSAG a new peak appears (tR: 5 min), corresponding to the more lipophilic Pba-GSGR. (c) No fluorescence emission is detected for the quenched prodrugs (here prodrug 4). (d) Enzymatic activation of the prodrugs (here prodrug 4) leads to release of the fluorescent Pba-GSGR (tR of 5 min. (e) Under the same conditions, the D-control 4* was not activated by uPA. 1574

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

Figure 3. (A, left) Fluorescence increase of prodrugs 1 (▲), 2 (▼), 3 (●), 4 (◆), D-control 4* (◇) and, 5 (□) incubated with PC-3M-luc-C6. (B, right) Fluorescence increase of 4 (◆), D-control 4* (◇) in uPA negative cell line LNCaP. All measurements were performed in sextuplicate and are expressed as mean values ± SD.

Figure 4. (A) Bioluminescence of luciferase-expressing PC-3M-luc-C6 tumor after D-luciferin peritoneal injection. (B) Tumor fluorescence intensity following administration of 2.0 mg/kg of prodrug 4 (as Pba equivalents). (C) Time-dependent fluorescence increase after systemic (retro-orbital) injection of 2.0 mg/kg Pba equiv of prodrug 1 (▲), 2 (▼), 3 (●), 4 (◆) and, 5 (□) and of (D) prodrug 4 (◆) and the corresponding D-control conjugate 4* (◇). Values are expressed as the mean ratio (Ft/F0 ± SEM) between the fluorescence emission at time t (Ft) and the autofluorescence (F0) in the respective tumor (n = 3).

activation of prodrug 4 in PC-3M-luc-C6 were significantly higher after 24 h than the differences observed in LNCaP cells. Therefore, we attributed the apparent activation of 4 and the corresponding D-control to a change in their configuration when interacting with cell membranes or other components present in the medium. In Vivo Fluorescence and Bioluminescence Imaging. To monitor noninvasively prodrug accumulation and activation in a subcutaneous PCa mouse model, prodrugs were administered systemically (2.0 mg/kg as Pba equivalents) and fluorescence and bioluminescence images were taken. As an example, Figure 4A,B shows an imaging sequence for prodrug 4. The image on the left corresponds to the bioluminescence produced by PC-3M-luc-C6 cancer cells after administration of luciferase allowing for the localization of tumor grafts. The other images show the fluorescence increase after injection of the prodrug as a function of time. Six hours after injection, the prodrug is largely accumulated in tumors and the liver, with a

Information). The activation of the higher pegylated version of the positively charged prodrug 2 was the least efficient of all prodrugs and 3 times lower at this time point as compared to the corresponding compound carrying only one PEG (1). Prodrug 3's fluorescence intensity increased by a factor of 6 when incubated with PCa cells for 46 h. Interestingly, the corresponding derivative with slightly longer side chains 4 showed only a 4-fold increase over this time period. Furthermore, the corresponding D-control showed also some increase in fluorescence intensity when incubated with PC-3Mluc-C6 cells. The difference between 4 and its D-control was statistically significant (P < 0.001) after 22 h of incubation. In order to test our compounds for some specificity for uPA, we have incubated the LNCaP cells, which do not express this protease,30 with prodrug 4 and its D-control (see Figure 3B). For both compounds spontaneous fluorescence increase over the first three hours followed by a continuous increase over the next 21 h was observed. However, the differences between 1575

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

Figure 5. (A) Tumoral fluorescence intensity following systemic (retro-orbital) administration of 5.0 mg/kg of prodrug 4 as Pba equivalents. (B) Bioluminescence in vivo imaging after PDT performed 16 h after administration. Fifteen minutes before imaging D-luciferin was administered peritoneally.

fluorescence increase of up to 5 times inside tumors compared to the fluorescence immediately after injection. Twenty-four hours after injection the fluorescence remains mainly confined to the tumors. After this time point, the fluorescence signal is 4 times higher in the tumor as compared to the adjacent tissue. Figure S4 in the Supporting Information compares the fluorescence of collected tumor, surrounding tissues and other organs. Tumor fluorescence over time of the different prodrugs is depicted in Figure 4C. All prodrugs show enhancement of fluorescence intensity during the first 6 h after administration, followed by a plateau. In contrast to the in vitro results, positively charged prodrugs 1, 2 and 5 showed lower fluorescence intensities compared to the neutral counterparts 3 and 4, which accumulated well in tumors (see also Figure S5 in the Supporting Information). Furthermore, the data also suggest a rapid clearance of the positively charged prodrugs as compared to the neutrally charged. No significant differences of total fluorescence (calculated as the surface area under the curve) were observed between prodrugs 1, 2, 3 and 5 (P > 0.05) and between prodrugs 3 and 4, respectively (P > 0.05). Prodrug 4 was statistically different from the positively charged prodrugs (P < 0.05). Figure 4D compares the profiles obtained after administration of prodrug 4 and its homologous D-control 4*. After administration of prodrug 4, activation by endogenous uPA led to a significantly increased fluorescence, whereas fluorescence of the D-control conjugate 4* remained closer to the baseline level (P < 0.05). PDT on PCa Xenografts. Bioluminescence in vivo imaging was used to assess the photodynamic effect on tumors after administration of prodrug 4 in vivo. Right tumors of mice injected systemically (retro-orbital injection) with 2.5, 5.0, and 10.0 mg/kg as Pba equiv were irradiated 16 h after prodrug administration with 100 J/cm2 at 665 nm. Left tumors were not irradiated and served as controls. Bioluminescence images were taken before irradiation as well as 24, 72, 120, and 168 h after PDT. Strong reductions of bioluminescence signal were observed in mice treated with 5.0 and 10.0 mg/kg doses of prodrug. Bioluminescent images after 24 h (see Figure 5) suggest a vast destruction of tumor cells in the irradiated tumor (right) as no bioluminescent signal is observed. Nonirradiated tumors are not affected. PDT related effects such edema and necrosis became visible 24 h after irradiation in the treated areas. While the bioluminescence intensity in the nontreated areas continued to increase over the observation period, in the treated areas a shallow signal became visible only 6 days in the periphery of the treated area.

strategies and therapeutic approaches.31−34 More recently this concept has also been translated into PDT, with the major goal of enhancing selectivity. We and others have tailored several protease-sensitive photodynamic agents to specific diseaseassociated proteases.22,23 We have recently successfully applied this strategy for the selective delivery of the photosensitizer to inflammatory lesions in an in vivo rheumatoid arthritis model.35 In the present study, we successfully designed a uPA-sensitive prodrug for simultaneous detection and treatment of PCa. In a previous study, we have already tried to optimize the carrier design in uPA-PPPs through backbone modifications in two prostate cancer cells lines in vitro with respect to water solubility, activation, and specificity for the target protease.28 This was done by modifying the backbone charge through the introduction of either negatively charged 2-sulfobenzoic acid or permanently positively charged moieties 4-imidazole acetic acid and N-methylnicotinic acid derivatives. Additionally in analogy to similar smart probes20 a neutrally charged compound substituted with 5 kDa mPEG moieties was prepared. This compound, however, displayed only low quenching, presumably due to hindering of PS−PS interaction. On the other hand, all three charged conjugates were nicely quenched, but only the positively charged compounds were sufficiently activated to exert a satisfying photodynamic effect on both cell lines in vitro. Furthermore, the negatively charged conjugate showed a high dark toxicity. We found that the positively charged prodrug 1 containing N-methylnicotinic acid as backbone modifying group had a good fluorescence quenching, ROS quenching and water solubility. This prodrug in combination with light proved to be efficient in killing PCa cells overexpressing uPA in vitro. This prodrug was designed to have a molecular weight that overpasses the threshold for renal filtration (30 to 50 kDa)36 and was pegylated to avoid the rapid elimination by the liver and components of the RES, which are particularly accelerated for charged polymers.37 Furthermore, pegylation with substitution of one 20 kDa mPEG seemed to increase the specificity of the uPA-PPP through compartmentalization since it avoided nonspecific activation by intracellular proteases. To our surprise, in contrast to similar probes for the targeting of proteases involved in rheumatoid arthritis, in a murine model of PCa, this compound showed suboptimal biodistribution with very low tumor accumulation probably due to its rapid elimination from the bloodstream. The key of our PPP approach is based not only on its accumulation in the target tissue but also on its selective proteolytic activation. We have used the luciferase transfected cell line PC-3M-Luc-C6 as the basis of our experimental animal model. These cells are stably transfected PC-3 M cells. Although there are no reports on uPA and uPAR levels on the transfected cells, PC-3 M cells have shown decreased secreted levels of uPA38 and significantly increased levels of uPAR39 compared to PC-3 cells. Intratumor injection of the

■

DISCUSSION Altered proteolytic activity in neoplastic and other degenerative diseases makes proteases attractive targets for disease imaging 1576

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

low fluorescence increase. In comparison, noncharged prodrugs 3 and 4 showed higher fluorescence intensities. An analogous thrombin-sensitive (TS) prodrug to prodrug 1 was also developed in our lab.35 Conversely, this TS-prodrug was highly accumulated and activated in arthritic lesions in CIA mice. The high accumulation and activation of the prodrug may be related to the physicochemical characteristics of the inflamed synovium and its high thrombin activity as well as the peptidyl substrate. We expected a higher tumor accumulation in xenografts when increasing molecular weight of the polymeric carrier. However, no significant effect was observed when the 45 kDa PL (prodrug 5) was used instead of the 18 kDa PL (prodrug 1) or when mPEG 20 kDa proportion (prodrugs 2 and 5) was increased. In contrast, changing the charge of the substituents from positive (prodrug 1) to neutral (prodrugs 3 and 4) significantly increased the accumulation in the tumor tissue. In this study, we have used the IVIS imaging system to monitor the fluorescence increase in tumors following the systemic administration of prodrugs of different designs. It has to be noted that this device cannot enable one to distinguish between the fluorescence emission of the intact prodrugs and photosensitizer fragments. Therefore, it is difficult to ascertain the exact contribution at a specific time point of (i) accumulation of nonactivated prodrug, (ii) specifically activated prodrug in the tumor, and (iii) nonspecifically activated prodrug in other organs followed by accumulation in the tumor. However, the contribution of site-specific proteolytic activation can be seen when comparing the differences in the fluorescence intensities of prodrug 4 and its D-control. Furthermore, by monitoring the fluorescence emission spectra in inflamed joints of CIA mice we are able to attribute the fluorescence observed in the target tissue to a nonquenched Pba fragment.35 Although nonspecific release of Pba fragments in distant organs and subsequent favored accumulation in the tumors such as observed for other PS42 are possible, this could be more the case for prodrug 3, which shows better in vitro activation than 4 but nearly the same accumulation as 4*. In vivo, uPA-PPPs are exposed to various enzyme activities, particularly after passive accumulation to certain lymphatic organs and to the liver. Here, prodrugs may be degraded nonspecifically, but due to the multiple selectivity of PDT, cytotoxicity can be confined through selective irradiation. PPPs are designed to label lesions characterized by enhanced proteolytic activity and to induce selective cell death following local irradiation. The protease-mediated phototoxicity of polymeric photosensitizer agents has been demonstrated in vitro and in vivo.22,23,43,44 The latter constitutes a proof-ofprinciple that tumor-associated proteases can activate polymeric compounds by cleavage of the polymeric backbone sufficiently to mediate phototoxicity. In contrast to these agents, in our design a protease-sensitive peptide linker is introduced between the PS and the polymeric backbone to increase the specificity of the prodrug to the target protease. In our preliminary PDT studies, we have demonstrated that it is feasible to design a uPA-PPP that enables the specific destruction of proliferating cells in vivo and to monitor the response and recurrence through bioluminescence imaging. Preliminary studies with the noncharged prodrug 4 in nude mice demonstrated the feasibility to eradicate PCa cells in vivo, as evidenced by complete disappearance of the tumor bioluminescence 24 h after irradiation. However, initial recurrences became apparent in most animals six days after PDT. In future studies, this should be optimized using different treatment conditions

positively charged prodrug 1, which was poorly accumulated in tumors after systemic administration, resulted in an 8.5-fold fluorescence intensity increase compared to the fluorescence immediately after injection (Figure S6 in the Supporting Information). This proves the enzymatic activity of the transfected PC-3M-Luc-C6 cells and underlines that the low fluorescent signal of tumors after systemic injection is primarily due to its suboptimal biodistribution and not due to a poor activation. Therefore, we modified the polymeric backbone of the positively charged uPA-PPP in order to optimize the pharmacokinetics, particularly the biodistribution without substantial loss of quenching capacity and solubility. Our modifications led to changes in the molecular size and also in the net charge of prodrugs. Basically, uPA-PPPs consisted of a polymeric backbone of 18 kDa or 45 kDa poly-L-lysine with a previously optimized Pba−peptide loading of 25% per PL chain and, pegylated with 20 kDa mPEG. The remaining ε-lysine residues were capped with either the positively charged Nsuccinimidyl (1-methyl-3-pyridinio) formate iodide or the neutral small mPEGs: mPEO4 and mPEO8. PPPs require efficient energy transfer between closely positioned photosensitizers on the polymeric backbone to depopulate irradiation-excited first and triplet states. This results in reduced fluorescence and energy transfer to molecular oxygen. It has been demonstrated that the fluorescence and ROS generation capacity of the lipophilic photosensitizer Pba can be efficiently quenched, if loaded at a sufficient amount on an appropriate polymeric backbone.29,40,41 In the present study, all backbone designs showed a reasonable quenching of fluorescence and ROS production. However, differences between prodrugs evidence that the increase in molecular size reduces their fluorescence while the degree of pegylation mainly alters the ROS production. Activation of protease-sensitive prodrugs occurs via specific disease related triggers which should ultimately result in higher selectivity. Therefore, another fundamental requirement for the appropriate performance of uPA-PPPs is the selective and efficient activation by uPA, the target protease. Using the reported minimal substrate GSGRSAG, prodrug activation occurred for all prodrugs efficiently by cleavage between the arginine-serine residues as confirmed by RP-HPLC and mass analysis of the released Pba-GSGR fragment. However, the in vitro activation by uPA was markedly reduced as the molecular size and/or degree of pegylation increased. This is in agreement with previous studies where the attachment of 20 kDa PEG or 5 kDa PEG chains to the polymeric backbone negatively affected enzymatic activation.28 Differences in enzymatic activation between various prodrugs suggest that enzyme− substrate interactions are not restricted to the active site and, therefore, prodrug activation may be explained also in terms of inter- and intramolecular interactions that result in steric hindrance from the reactive site. Activation of prodrugs by uPa in the test tube and by PCa cells showed contrasting results. The exogenous proteases activated more efficiently the cationic prodrugs (1 and 2) than their neutral counterparts (3 and 4) whereas incubation of prodrugs with cells showed no such tendency. However, specific activation of uPA-sensitive prodrugs was demonstrated using a control sequence built of the corresponding D-amino acids and a cell line void of uPA expression. Following systemic administration, cationic prodrugs 1, 2 and 5 accumulated only slightly in tumors, as evidenced by the 1577

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

ROS, reactive oxygen species; ROIs, regions of interests; uPA, urokinase-like plasminogen activator

including higher drug and light doses or through the use of multiple dosing/PDT schedules and observation of animals over a longer time period.

■

■

CONCLUSIONS We developed a uPA sensitive agent that fluorescently labels and eradicates PCa (cells) in vivo after localized light irradiation. This prodrug is capable of accumulating substantially in a murine xenograft model for PCa. It is locally activated by upregulated uPA, selectively releasing the photosensitizer Pba in tumor tissue. Finally, it induces eradication of cancer cells after irradiation of fluorescent tumors. Unfavorable PS biodistribution and insufficient selectivity toward pathological tissue are recognized as major limitations of PDT of PCa, frequently causing skin photosensitization and extraprostatic treatment effects. Our strategy combining passive targeting of tumors due to the EPR effect and site-selective PS release, localized light irradiation and phototoxicity could increase selectivity of PDT for the treatment of PCa.

■

(1) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M. J. Cancer statistics, 2008. CA Cancer J. Clin. 2008, 58 (2), 71−96. (2) Denmeade, S. R.; Isaacs, J. T. A history of prostate cancer treatment. Nat. Rev. Cancer 2002, 2 (5), 389−96. (3) Makarov, D. V.; Loeb, S.; Getzenberg, R. H.; Partin, A. W. Biomarkers for prostate cancer. Annu. Rev. Med. 2009, 60, 139−51. (4) Pavlou, M.; Diamandis, E. P. The search for new prostate cancer biomarkers continues. Clin. Chem. 2009, 55 (7), 1277−9. (5) Parker, C.; Muston, D.; Melia, J.; Moss, S.; Dearnaley, D. A model of the natural history of screen-detected prostate cancer, and the effect of radical treatment on overall survival. Br. J. Cancer 2006, 94 (10), 1361−8. (6) Moore, C. M.; Pendse, D.; Emberton, M. Photodynamic therapy for prostate cancera review of current status and future promise. Nat. Clin. Pract. Urol. 2009, 6 (1), 18−30. (7) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3 (5), 380−7. (8) Muschter, R. Photodynamic therapy: a new approach to prostate cancer. Curr. Urol. Rep. 2003, 4 (3), 221−8. (9) Windahl, T.; Andersson, S. O.; Lofgren, L. Photodynamic therapy of localised prostatic cancer. Lancet 1990, 336 (8723), 1139. (10) Nathan, T. R.; Whitelaw, D. E.; Chang, S. C.; Lees, W. R.; Ripley, P. M.; Payne, H.; Jones, L.; Parkinson, M. C.; Emberton, M.; Gillams, A. R.; Mundy, A. R.; Bown, S. G. Photodynamic therapy for prostate cancer recurrence after radiotherapy: a phase I study. J. Urol. 2002, 168 (4 Part 1), 1427−32. (11) Moore, C. M.; Nathan, T. R.; Lees, W. R.; Mosse, C. A.; Freeman, A.; Emberton, M.; Bown, S. G. Photodynamic therapy using meso tetra hydroxy phenyl chlorin (mTHPC) in early prostate cancer. Lasers Surg. Med. 2006, 38 (5), 356−63. (12) Zaak, D.; Sroka, R.; Khoder, W.; Adam, C.; Tritschler, S.; Karl, A.; Reich, O.; Knuechel, R.; Baumgartner, R.; Tilki, D.; Popken, G.; Hofstetter, A.; Stief, C. G. Photodynamic diagnosis of prostate cancer using 5-aminolevulinic acid--first clinical experiences. Urology 2008, 72 (2), 345−8. (13) Pinthus, J. H.; Bogaards, A.; Weersink, R.; Wilson, B. C.; Trachtenberg, J. Photodynamic therapy for urological malignancies: past to current approaches. J. Urol. 2006, 175 (4), 1201−7. (14) Verigos, K.; Stripp, D. C.; Mick, R.; Zhu, T. C.; Whittington, R.; Smith, D.; Dimofte, A.; Finlay, J.; Busch, T. M.; Tochner, Z. A.; Malkowicz, S.; Glatstein, E.; Hahn, S. M. Updated results of a phase I trial of motexafin lutetium-mediated interstitial photodynamic therapy in patients with locally recurrent prostate cancer. J. Environ. Pathol., Toxicol. Oncol. 2006, 25 (1−2), 373−87. (15) Patel, H.; Mick, R.; Finlay, J.; Zhu, T. C.; Rickter, E.; Cengel, K. A.; Malkowicz, S. B.; Hahn, S. M.; Busch, T. M. Motexafin lutetiumphotodynamic therapy of prostate cancer: short- and long-term effects on prostate-specific antigen. Clin. Cancer Res. 2008, 14 (15), 4869−76. (16) Weersink, R. A.; Bogaards, A.; Gertner, M.; Davidson, S. R.; Zhang, K.; Netchev, G.; Trachtenberg, J.; Wilson, B. C. Techniques for delivery and monitoring of TOOKAD (WST09)-mediated photodynamic therapy of the prostate: clinical experience and practicalities. J. Photochem. Photobiol., B 2005, 79 (3), 211−22. (17) Trachtenberg, J.; Bogaards, A.; Weersink, R. A.; Haider, M. A.; Evans, A.; McCluskey, S. A.; Scherz, A.; Gertner, M. R.; Yue, C.; Appu, S.; Aprikian, A.; Savard, J.; Wilson, B. C.; Elhilali, M. Vascular targeted photodynamic therapy with palladium-bacteriopheophorbide photosensitizer for recurrent prostate cancer following definitive radiation therapy: assessment of safety and treatment response. J. Urol. 2007, 178 (5), 1974−9. (18) Trachtenberg, J.; Weersink, R. A.; Davidson, S. R.; Haider, M. A.; Bogaards, A.; Gertner, M. R.; Evans, A.; Scherz, A.; Savard, J.; Chin, J. L.; Wilson, B. C.; Elhilali, M. Vascular-targeted photodynamic therapy (padoporfin, WST09) for recurrent prostate cancer after

ASSOCIATED CONTENT

S Supporting Information *

Figures depicting analytical HPLC traces, SEC-MALLS-RI-UV analysis, fluorescence−time profile, and fluorescence intensity; table of comparison of fluorescence and ROS quenching factors. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland. Phone: +41 22 37 933 35. Fax: +41 22 37 965 67. E-mail: [email protected]. Present Address †

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02139 Massachusetts, USA, and Department of Anesthesiology, Division of Critical Care, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank the Swiss National Science Foundation for their kind support. This work is supported by Grants 205320-122144, 205321_126834, 205320_138309, K32K1-116460, CR32I3_129987, and IZLSZ2_123011. D.G. is supported by SNF Grant PBGEP3-129111.

■

ABBREVIATIONS USED NICO, 1-methyl-3-pyridinio formate iodide; ACN, acetonitrile; DCM, dichloromethane; TFA, diethyl ether and trifluoroacetic acid; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EPR, enhanced permeability and retention; FBS, fetal bovine serum; FOV, field of view; DIPEA, N,N-diisopropylethylamine; mPEO8, methyl octaethylene oxide; mPEG20, methyl polyethylene glycol 20 kDa; PEO4, methyl tetraethylene oxide; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate; Pba, pheophorbide a; PDT, photodynamic therapy; PS, photosensitizer; PL, poly lysine; PPP, polymeric photosensitizer prodrug; PCa, prostate cancer; 1578

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579

Molecular Pharmaceutics

Article

failure of external beam radiotherapy: a study of escalating light doses. Brit. J. Urol. Int. 2008, 102 (5), 556−62. (19) Haider, M. A.; Chung, P.; Sweet, J.; Toi, A.; Jhaveri, K.; Menard, C.; Warde, P.; Trachtenberg, J.; Lockwood, G.; Milosevic, M. Dynamic contrast-enhanced magnetic resonance imaging for localization of recurrent prostate cancer after external beam radiotherapy. Int. J. Radiat. Oncol., Biol., Phys. 2008, 70 (2), 425−30. (20) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 2000, 65 (1−2), 271−84. (21) Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov, A., Jr. In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 1999, 17 (4), 375−8. (22) Choi, Y.; Weissleder, R.; Tung, C. H. Protease-mediated phototoxicity of a polylysine-chlorin(E6) conjugate. ChemMedChem 2006, 1 (7), 698−701. (23) Campo, M. A.; Gabriel, D.; Kucera, P.; Gurny, R.; Lange, N. Polymeric photosensitizer prodrugs for photodynamic therapy. Photochem. Photobiol. 2007, 83 (4), 958−65. (24) Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G. Activatable photosensitizers for imaging and therapy. Chem. Rev. 2010, 110 (5), 2839−57. (25) Cozzi, P. J.; Wang, J.; Delprado, W.; Madigan, M. C.; Fairy, S.; Russell, P. J.; Li, Y. Evaluation of urokinase plasminogen activator and its receptor in different grades of human prostate cancer. Hum. Pathol. 2006, 37 (11), 1442−51. (26) Shariat, S. F.; Roehrborn, C. G.; McConnell, J. D.; Park, S.; Alam, N.; Wheeler, T. M.; Slawin, K. M. Association of the circulating levels of the urokinase system of plasminogen activation with the presence of prostate cancer and invasion, progression, and metastasis. J. Clin. Oncol. 2007, 25 (4), 349−55. (27) Ke, S. H.; Coombs, G. S.; Tachias, K.; Navre, M.; Corey, D. R.; Madison, E. L. Distinguishing the specificities of closely related proteases. Role of P3 in substrate and inhibitor discrimination between tissue-type plasminogen activator and urokinase. J. Biol. Chem. 1997, 272 (26), 16603−9. (28) Gabriel, D.; Zuluaga, M. F.; Martinez, M. N.; Campo, M. A.; Lange, N. Urokinase-plasminogen-activator sensitive polymeric photosensitizer prodrugs: design, synthesis and in vitro evaluation. J. Drug Delivery Sci. Technol. 2009, 19 (1), 15−24. (29) Gabriel, D.; Campo, M. A.; Gurny, R.; Lange, N. Tailoring protease-sensitive photodynamic agents to specific disease-associated enzymes. Bioconjugate Chem. 2007, 18 (4), 1070−7. (30) Pentyala, S. N.; Whyard, T. C.; Waltzer, W. C.; Meek, A. G.; Hod, Y. Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix. Cancer Lett. 1998, 130 (1−2), 121−6. (31) Weissleder, R.; Cheng, H. C.; Marecos, E.; Kwong, K.; Bogdanov, A. Non-invasive in vivo mapping of tumour vascular and interstitial volume fractions. Eur. J. Cancer 1998, 34 (9), 1448−54. (32) Tung, C. H.; Mahmood, U.; Bredow, S.; Weissleder, R. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000, 60 (17), 4953−8. (33) Law, B.; Curino, A.; Bugge, T. H.; Weissleder, R.; Tung, C. H. Design, synthesis, and characterization of urokinase plasminogenactivator-sensitive near-infrared reporter. Chem. Biol. 2004, 11 (1), 99−106. (34) Gabriel, D.; Zuluaga, M. F.; van den Bergh, H.; Gurny, R.; Lange, N. It is all about proteases: from drug delivery to in vivo imaging and photomedicine. Curr. Med. Chem. 2011, 18 (12), 1785− 805. (35) Gabriel, D.; Busso, N.; So, A.; van den Bergh, H.; Gurny, R.; Lange, N. Thrombin-sensitive photodynamic agents: A novel strategy for selective synovectomy in rheumatoid arthritis. J. Controlled Release 2009, 138 (3), 225−34. (36) Fox, M. E.; Szoka, F. C.; Frechet, J. M. Soluble polymer carriers for the treatment of cancer: the importance of molecular architecture. Acc. Chem. Res. 2009, 42 (8), 1141−51.

(37) Owens, D. E., 3rd; Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307 (1), 93−102. (38) Sehgal, I.; Forbes, K.; Webb, M. A. Reduced secretion of MMPs, plasminogen activators and TIMPS from prostate cancer cells derived by repeated metastasis. Anticancer Res. 2003, 23 (1A), 39−42. (39) Forbes, K.; Gillette, K.; Kelley, L. A.; Sehgal, I. Increased levels of urokinase plasminogen activator receptor in prostate cancer cells derived from repeated metastasis. World J. Urol. 2004, 22 (1), 67−71. (40) Hackbarth, S.; Horneffer, V.; Wiehe, A.; Hillenkamp, F.; Roder, B. Photophysical properties of pheophorbide-a-substituted diaminobutane poly-propylene-imine dendrimer. Chem. Phys. 2001, 269 (1− 3), 339−46. (41) Hackbarth, S.; Ermilov, E. A.; Roder, B. Interaction of Pheophorbide a molecules covalently linked to DAB dendrimers. Opt. Commun. 2005, 248 (1−3), 295−306. (42) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. Photodynamic therapy. J. Natl. Cancer Inst. 1998, 90 (12), 889−905. (43) Soukos, N. S.; Hamblin, M. R.; Hasan, T. The effect of charge on cellular uptake and phototoxicity of polylysine chlorin(e6) conjugates. Photochem. Photobiol. 1997, 65 (4), 723−9. (44) Choi, Y.; Weissleder, R.; Tung, C. H. Selective antitumor effect of novel protease-mediated photodynamic agent. Cancer Res. 2006, 66 (14), 7225−9.

1579

dx.doi.org/10.1021/mp2005774 | Mol. Pharmaceutics 2012, 9, 1570−1579