Intracellular Cargo Delivery by an Octaarginine Transporter Adapted

Bioconjugate Chem. 2006, 17, 787−796

787

Intracellular Cargo Delivery by an Octaarginine Transporter Adapted to Target Prostate Cancer Cells through Cell Surface Protease Activation Elena A. Goun,† Rajesh Shinde,‡ Karen W. Dehnert,† Angie Adams-Bond,† Paul A. Wender,† Christopher H. Contag,‡ and Benjamin L. Franc*,§ Departments of Chemistry and Molecular Pharmacology, Stanford University, Stanford, California 94305, Departments of Pediatrics, Microbiology, and Immunology, and Radiology, Stanford University, Stanford, California 94305, and Department of Radiology and Bioengineering Program, University of California, San Francisco, 505 Parnassus Avenue, San Francisco, California 94143-0252. Received November 7, 2005; Revised Manuscript Received February 1, 2006

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the D-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

INTRODUCTION Targeted delivery systems are critical for refining effective molecular imaging approaches and enhancing therapeutic intervention by achieving tissue-specific localization. In oncology, a number of tumor-associated molecules have been identified, and many of these have enzymatic functions that can be exploited for cancer imaging and treatment. Prostate cancer, for example, expresses PSA, a cancer-specific enzyme that can be utilized for prodrug applications. Currently, there is no treatment that significantly prolongs survival in the setting of metastatic prostate cancer (1). Androgen ablation therapy, although of substantial palliative benefit, has little impact on overall survival, and the mortality rate from prostate cancer continues to increase (2, 3). The low proliferation rate of androgen-independent prostate cancer requires that standard antiproliferative chemotherapeutic agents be administered over prolonged periods risking induction of dose-limiting toxicities (4, 5). Likewise, no single imaging strategy has been identified as ideal for prostate cancer at this time. Imaging modalities that rely on anatomic changes in tissue (e.g., computed tomography, CT) lack sensitivity for small tumor-containing lymph nodes and lack selectivity for large tumor-free nodes merely reacting to local inflammation. Therefore, there has been much emphasis on the development of biologically based imaging techniques * To whom correspondence should be addressed. Mailing address: Benjamin Franc, M. D., Department of Radiology, University of California, San Francisco, 505 Parnassus Avenue, L340, San Francisco, CA 94143-0252. E-mail: [email protected]. Phone: (415) 353-4219. Fax: (415) 353-8571. † Departments of Chemistry and Molecular Pharmacology, Stanford University. ‡ Departments of Pediatrics, Microbiology, and Immunology, and Radiology, Stanford University. § University of California, San Francisco.

such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) because they have the potential to more accurately assess nodal disease. The low metabolic rate of prostate cancer provides little uptake of the most widely utilized PET radiopharmaceutical, the glucose analogue 18F-fluoro-deoxy-glucose (FDG). A few radiopharmaceuticals exploiting other molecular pathways, such as 11Cacetate, have demonstrated higher levels of uptake within prostate cancer than FDG (6). 11C-Acetate and its fluorinated counterpart, 18F-fluoroacetate, are promising imaging agents for detection of prostate cancer recurrence (7, 8). 11C-Choline and its fluorinated counterpart, 18F-fluorocholine, have also demonstrated greater uptake in prostate cancer than FDG and may be particularly useful in identifying osseous and lymph node metastases (9-11). While the relationship between their molecular target and their potential role in the choice of therapeutic management is clearer than many of the other radiopharmaceuticals, the radiolabeled androgens have not shown as high a sensitivity for prostate cancer in initial investigations (12-14). Gamma-emitting radiopharmaceuticals for SPECT, such as indium-111 (111In)-radiolabeled prostate specific membrane antigen (PSMA) monoclonal antibody, have demonstrated challenges such as long blood clearance and localization times, nonspecific localization, redistribution, and poor tissue penetration (15-17). Although many of the molecular methods currently employed in prostate cancer imaging and therapy utilize a mechanism to concentrate the delivery molecule in the cancer cell, these concentration mechanisms are often tied to a metabolic pathway that has a finite transporting capacity. Clearly, it would be useful to develop other strategies to deliver cytotoxic therapeutics or imaging agents specifically to prostate cancer cells, and exploitation of cell-selective enzymes such as PSA opens the door to unique mechanisms of delivery. Cell-specific targeting based on protease expression remains a challenge, and intracellular delivery appears to be a key in several approaches to

10.1021/bc0503216 CCC: $33.50 © 2006 American Chemical Society Published on Web 04/28/2006

788 Bioconjugate Chem., Vol. 17, No. 3, 2006

Goun et al. Scheme 1. Proposed Mechanism for Selective Uptake of APTDs in PSA-Expressing Prostate Cancer Cellsa

Figure 1. HPLC analysis of proteolysis of APTD by PSA. The presence of peptide 19 was plotted versus time following addition of active PSA and incubation at 37 °C giving t1/2 ) 123 min (∼2 h).

imaging and therapy. Protein transduction domains (PTDs) or cell-penetrating peptides (CPPs) and more generally molecular transporters (MTs) are molecules that can be conjugated to a cargo to enable its entry as a conjugate into cells (18-20). An example of such a molecule in this rapidly emerging field of molecular transporters is the highly basic region (Tat49-57, RKKRRQRRR) of the nuclear transcription activator protein (Tat) encoded by human immunodeficiency virus type 1 (HIV1) (21-23). Our previous structure-function studies of the TAT nonamer have demonstrated that D-oligoarginine peptides as well as spaced oligoarginine peptides (rXrXrXrXrXrXr), guanidinerich peptoids with 1,4-spaced side chains, guanidinylated dendrimers, and guanidinylated oligocarbamates are superior to Tat49-57 in cellular uptake and offer significant cost and availability advantages (24-28). Conjugates of these transporters and various probes (e.g., fluorescein) or drugs (e.g., cyclosporin A) are highly water soluble and rapidly enter cells and tissues (25, 28-30), enabling their advancement into human trials (25). Cargoes ranging in size from metal chelates and fluorescent dyes (31, 32) to iron oxide nanoparticles (33), proteins (30), and liposomes (34) can be transported into cells. Based on a previously published mechanistic theory (35) of oligoarginine translocation through cell membranes, we hypothesized that conjugates in which negatively charged residues are covalently attached to an r8 PTD would enter cells less readily, if at all. We therefore developed protransporter agents for PSA-mediated prostate cancer targeting in which r8 PTDcontaining peptides would be inactivated for cellular uptake through intramolecular complexation with attached polyanionic residues. This inactivated form would be expected to distribute systemically but would subsequently be activated for cellular uptake by PSA-mediated proteolytic cleavage at the site of prostate cancer metastases. To produce an “activatable” protein transduction domain (Figure 1, Scheme 1; APTD), an r8 PTD would be attached to an attenuating domain (C) through a cleavable linker (CHSSKLQG) (B), a well-studied sequence selected for its balance of enzyme selectivity and cleaving kinetics (3, 36, 41-42). Intramolecular association of residues in C with the r8 PTD would disfavor intermolecular association of the r8 PTD with cell surface moieties and thereby disfavor uptake by the translocation mechanism. Tissue-selective proteolytic cleavage of B by prostate specific antigen (PSA), a protease associated with the extracellular membrane of prostate cancer cells, would eliminate internal inactivation of r8 PTD by C, thereby enabling r8 PTD-directed cellular entry. Together, these three components make up a complete APTD that remains

a (i) Intact APTD conjugate with r PTD (A), PSA-protease-specific 8 substrate linkage (B), and r8 PTD-attenuating sequence regions (C). (ii) Upon encountering PSA associated with the surface of a prostate cancer cell, the PSA-specific substrate (B) is cleaved. (iii) Cleavage of APTD in step ii allows the fluorophor cargo-carrying r8 PTD to enter the prostate cancer cell while the r8 PTD-attenuating sequence remains in circulation.

extracellular until the specific peptide link is cleaved by a PSA (37-39). A similar approach based on a different protease has been recently utilized by another group to deliver imaging fluorophores into cells and squamous cell cancer xenografts in mice (40). Herein, we describe the design, synthesis, and characterization of “activatable” PTDs (APTDs) that localize in cells after cleavage by PSA. The r8 PTD-attenuating sequences were designed to disable the transmembrane transporting ability of the r8 PTD. A variety of r8 PTD-attenuating sequences combining charge-complementing aspartic acid residues spaced with varying numbers of glycine residues were developed. Because the critically important requirements for the attenuation component of the APTD and proteolytic release were unknown, a library of attenuated r8 PTD peptide molecules was synthesized and the efficiency of r8 PTD transport attenuation of various sequences was studied. These molecules were found to be cleaved by PSA and subsequently transported into Jurkat and PC3M prostate cancer cell lines. These results provide a new strategy for selective delivery of agents into prostate cancer cells and represent a new opportunity for targeted delivery of therapeutic and imaging agents based on r8 PTDs.

EXPERIMENTAL PROCEDURES General Methods. Unless otherwise stated, all reagents and solvents were obtained from commercial sources and used without further purification. Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed with a Varian ProStar 210/215 preparative HPLC using Alltec Alltima C18, 250 mm × 22 mm, column or analytical RP-HPLC (HewlettPackard with Agilent 1100 series automated injector) with analytical Zorbax SB C18, 150 mm × 4.6 mm, column. The products were eluted utilizing a solvent gradient (solvent A ) 0.1% trifluoroacetic acid (TFA)/H2O; solvent B ) 0.1% TFA/ CH3CN). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis was carried out using a Voyager-DE PRO biospectrometry workstation (PerSpective Biosystem Inc.). Ten microliters of each HPLC fraction was mixed with 20 µL of matrix solution (R-cyano-4hydroxycinnamic acid) in 60% acetonitrile/0.1% trifluoroacetic acid/water solution. The spectra were acquired in the linear

Intracellular Cargo Delivery by an Arg8 Transporter

mode, which, in general, provides values a few daltons higher than the monoisotopic mass values. Fluorescent images were obtained using an IVIS 50 imaging system (Xenogen Corp., Alameda, CA), which consists of a cooled CCD camera detector system incorporating a xenon light source and appropriate filters for excitation and emission wavelengths. The images were acquired and processed using LivingImage 2.50 software. Flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ) was performed using standard FITC filters. Automated Peptide Synthesis (Peptides 1-4). All reagents for peptide synthesis including N-methylpyrrolidone (NMP), diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), 1-hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU), and piperidine were purchased from Aldrich, NovaBiochem (CA), BaChem (CA), or Applied Biosystems (CA). Fmoc-protected amino acids and resins were purchased from NovaBachem or BaChem in their appropriately protected form. All automated peptide syntheses were performed on a PE Biosystems model 433A automated peptide synthesizer using the standard FastMoc coupling strategy. The peptide was assembled on Fmoc-Rink amide resin on a 0.1 or 0.25 mmol scale with all amino acids used in 4- to 10-fold excess (1 mmol). The purity of all peptides and peptide conjugates was established as a single peak by analytical HPLC (HewlettPackard with Agilent 1100 series automated injector) with an analytical Zorbax SB C18, 150 mm × 4.6 mm, column at a flow rate of 1.0 mL/min in the indicated acetonitrile/water buffer containing 0.1% trifluoroacetic acid using 5-45% gradient over 15 min followed by 5 min wash at 100% acetonitrile buffer. The identity of all peptides and peptide conjugates was established using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Manual Peptide Synthesis. Fmoc-Rink amide resin was mixed in DMF under nitrogen in a fritted peptide vessel (20 min). The Fmoc-protecting group was removed by mixing resin in a 20% piperidine/DMF solution (10 mL/g of resin) for 15 min (2×). The vessel was drained via vacuum and washed with DMF (3×). The chloranil resin test was performed by adding one drop of 2% acetaldehyde in DMF followed by one drop of 2% p-chloranil in DMF to the resin with a positive test (indicating a free amine) resulting in blue-stained beads. A small sample of resin also underwent the Kaiser resin test where two drops each of (i) 5 g of ninhydrin in 100 mL ethanol, (ii) 80 g of liquefied phenol in 20 mL of ethanol, and (iii) 0.001 M aqueous sodium cyanide (2 mL) in 98 mL of pyridine were added and the mixture was shaken to mix and heated to 120 °C for 4 min. A positive test (indicating a free primary amine) resulted in blue-colored beads. Deprotection was repeated until the chloranil or Kaiser resin test was positive. The Fmoc-protected amino acid (5 equiv), HOBt (5 equiv), and HBTU (5 equiv) were dissolved in DMF (5 mL/g of resin) followed by the addition of DIEA (10 equiv). This mixture was added to the resin and agitated gently for 3060 min or until a negative resin test was obtained. After coupling, the resin was washed with DMF (3×). This Fmoc deprotection and coupling sequence was repeated until the desired peptide was assembled. Fluorescein Labeling. The fluorescent tag was attached through either the free amine of the N-terminus or a lysine side chain of the fully protected peptide while still attached to the solid support. The resin (0.1 mmol) was suspended in dry DMF in a polypropylene tube. Diisopropyl ethylamine (0.17 mL, 1 mmol, 10 equiv) and fluorescein isothiocyanate (200 mg, 0.5 mmol, 5 equiv) were added in one portion to the mixture. The solution was agitated for 18 h followed by vacuum

Bioconjugate Chem., Vol. 17, No. 3, 2006 789

filtration for removal of solvent. The resin was washed twice with DMF and once with DCM to remove any residual base and reagent. Deprotection and Cleavage of Peptides. The peptide-bound resin was washed well with DCM and dried under nitrogen for several hours. The peptide was deprotected and cleaved from the solid support by exposing the resin to a solution (10 mL/ 0.1 mmol of peptide) of 95% TFA and 5% triisopropylsilane (TIS) for non-cysteine-containing peptides or a solution of 94% TFA, 2.5% water, 2.5% 1,2-ethane dithiol (EDT), and 1% triisopropylsilane (TIS) for cysteine-containing peptides. The mixture was placed in a polypropylene tube and mixed for 24 h at ambient temperature. After 24 h, the solution was filtered and concentrated under reduced pressure to provide a crude oil. The oil was dissolved in a minimal volume of TFA, placed in a centrifuge tube and triturated with diethyl ether. The material was pelleted via centrifugation, and the ether was removed by decanting. The pellet was washed two additional times with dry diethyl ether and dried under nitrogen. The crude peptide was dissolved in 0.1% TFA/water then purified by reverse-phase HPLC (C18 packed column; water-acetonitrile, 0.1% TFA v/v) to afford a solid after lyophilization. Synthesis of Attenuating Sequences (Peptides 5-11). The Fmoc-Asp(OtBu)-OH or Fmoc-Gly-OH residues or both were manually attached to the resin as described above. The BocCys(NPys)-OH residue was coupled using N,N′-diisopropylcarbodiimide (DIC) (5 equiv) and HOBt (6 equiv) in 80:20 DMF/dichloromethane (DCM) (5 mL). The DIEA coupling strategy could not be employed due to the base-labile nature of the 3-nitro-2-pyridinesulfenyl (Npys) protecting group. After 16 h, the peptide was washed with DMF (3×) and DCM (3×) and dried under argon for several hours. Side-chain deprotection and cleavage from the resin was achieved by mixing the resinbound peptide in a 95:5 TFA/triisopropylsilane (TIS) solution for 5 h. Washing and purification was performed as described above. Synthesis of the CHSSKLQG-r8-K-(FITC)NH2 Conjugate (12). The peptide was assembled manually via solid-phase peptide synthesis on a 0.1 mmol scale using the coupling strategy described above. Amino acids Fmoc-Lys(Mtt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gln(tBu)-OH, FmocLeu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-His(Trt)-OH, and Fmoc-Cys(Trt)-OH (NovaBiochem, CA) were used in 4-fold excess (0.4 mmol). A solution of acetic anhydride (100 µL) and DIEA (350 µL) in DMF (2 mL) was then added, and the mixture was sparged with nitrogen gas for 1 h or until the chloranil resin test (see above) was negative indicating N-terminal acetylation. The resin was filtered via vacuum and washed with DMF (2 mL, 3×) and DCM (2 mL, 3×). A solution of TFA (1%) and TIS (3%) in DCM (96%) was then added, and the mixture was agitated under a nitrogen atmosphere for 5 min. This was repeated until a positive resin test was obtained (5×) indicating removal of the Mtt protecting group. To the free amine of the lysine side chain was added the fluorescein label according to the general procedure described above. The resin was dried for several hours under nitrogen before deprotection and cleavage (see general procedure above). Orange solid (189.7 mg, 36% yield). Analytical RP-HPLC (0.1% TFA in H2O/CH3CN): >98% purity. MALDI MS: calculated for C111H180N48O26S2 (M + H), 2667.05; found, 2667.10. Synthesis of the Final Attenuated Peptide Conjugates (APTD Compounds 13-19). To the attenuating peptide sequences (5-11) (4 µmol) in 0.1% TFA-water (0.5 mL) was added a solution of peptide 12 (5.3 mg, 2 µmol) in 0.1% TFAwater (1 mL). The mixture was stirred at room temperature for 24 h before quenching with ethyl acetate (2 mL). The layers were separated, and the aqueous phase was extracted and


Goun et al.

Table 1. Library of Synthesized Peptides peptide sequencea

MW, calcdb

MW, foundb

no.

1

r8-K(FITC)NH2

C75H123N35O14S 1771.07

1770.11

11c

CDGGDGGDGGD

2

r7-K(FITC)NH2

C69H111N31O13S 1614.88

1615.87

12

CHSSKLQG-r8-K(FITC)NH2

3

r5-K(FITC)NH2

C57H87N23O11S 1302.51

1302.01

13

DCCHSSKLQG-r8-K(FITC)NH2

4

r4-K(FITC)NH2

C51H75N19O10S 1146.33

1145.29

14

DDCCHSSKLQG-r8-K(FITC)NH2

5c

CD

390.46

15

DGDCCHSSKLQG-r8-K(FITC)NH2

6c

CDD

505.26

16

DGGDCCHSSKLQG-r8K(FITC)NH2

7c

CDGD

562.79

17

DGGGDCCHSSKLQG-r8K(FITC)NH2

8c

CDGGD

620.03

18

DGGDGGDCCHSSKLQG-r8K(FITC)NH2

9c

CDGGGD

675.90

19

DGGDGGDGGDCCHSSKLQG-r8K(FITC)NH2

10c

CDGGDGGD

C12H15N5O6S2, (M + H) 389.41 C16H20N6O9S2, (M + H) 504.49 C18H23N7O10S2, (M + H) 561.55 C20H6N8O11S2, (M + H) 618.60 C22H29N9O12S2, (M + H) 675.65 C28H37N11O16S2 (M + H) 847.79

no.

peptide sequencea

MW, calcdb

MW, foundb

C36H48N14O21S2 (M + H) 1076.98 C111H180N48O26S2 (M + H) 2667.05 C118H191N51O30S3 (M + H) 2900.30 C122H196N52O33S3 (M + H) 3015.39 C124H199N53O34S3 (M + H) 3072.44 C126H202N54O35S3 (M + H) 3129.49 C128H205N55O36S3 (M + H) 3186.54 C134H213N57O40S3 (M + H) 3358.68 C143H236N59O45S3 (M + H) 3597.96

1076.69 2667.10 2893.29 3014.96 3072.90 3129.91 3187.19 3359.76 3597.12

848.93

a FITC ) fluorescein isothiocyanate; r ) arginine octamer; all other letters correspond to standard single-letter abbreviations for amino acids. b Without 8 counter ions. c 3-Nitro-2-pyridinesulfenyl (Npys)-protected cysteine has been used for synthesis of peptides 5-11.

concentrated in vacuo. The APTD conjugated peptide was purified by RP-HPLC to give the desired conjugated peptide as a bright yellow sticky solid (90-95% yield). Analytical RPHPLC (0.1% TFA in H2O/CH3N): >99% purity. MALDI MS: see Table 1. Serum Stability Studies. The serum stability of APTD conjugates was evaluated by incubation in fetal calf serum (FCS) (50% FCS/50% phosphate-buffered saline (PBS), pH 7.4) at 37 °C for 4 h using a standard protocol (47, 48). The percentage of intact APTD peptide after incubation was determined by precipitation of the serum proteins with 70% acetonitrile in 0.1% TFA and separating degradation products by analytical RPHPLC using a 15 min, 10-30% linear acetonitrile gradient (0.1% TFA/water). Kinetic Analysis of Substrate Hydrolysis by PSA. The activity of human PSA protease (Calbiochem) was tested prior to experiments with a PSA substrate fluorogenic test kit (MuHis-Ser-Ser-Lys-Leu-Gln-AFC, 2TFA (Mu ) 4-morpholinoureidyl); Calbiochem). Subsequently, 100 µL of 50 µM PSA substrate solution in PSA buffer (50 mM Tris, pH 7.5, 150 mM NaCl) was added to 5.4 µL (concentration ) 1.84 mg/mL) of enzymatically active PSA solution in the same buffer, and the mixture was incubated at 37 °C for 4 h. Buffer without PSA enzyme was used as control. Progress of the enzymatic cleavage was monitored by reverse-phase analytical HPLC. Aliquots were drawn at specific times from 0 to 4 h. Ten microliters of the sample was diluted with 90 µL of 0.1% TFA to yield pH ≈ 2. After filtration (0.2 µM membrane filter, Anatop Laboratory), 80 µL of the resulting samples were applied to an analytical HPLC column using a 15 min, 5-30% linear acetonitrile gradient (0.1% TFA/water). Fractions were collected manually in Eppendorf tubes (1.5 mL) and dried completely, and masses were analyzed by mass spectroscopy (MALDI). Cell Uptake Assay: Jurkat Cells. The synthesized APTDs were each dissolved in PBS buffer (pH 7.2), and their concentrations were determined spectrophotometrically by

absorption of fluorescein at 490 nm ( ) 67 000) (21). Varying concentrations (50 and 0.4 µM) of each peptide (nonattenuated CHSSKLQG-r8-K(FITC)NH2 (12, see Table 1) and all attenuated APTD forms) were added to 3 × 106 Jurkat cells (human T cell line, grown in 10% FCS and DMEM) for 5 min in microtiter plates (96 well) at 37 °C. Peptides r5-K(FITC)NH2 (3) and r4-K(FITC)NH2 (4) were selected as controls. Previous work has shown that a minimum of six arginine residues is required for significant cell uptake to occur (25). Following incubation, the microtiter plates were centrifuged, and the cells were isolated, washed, and resuspended in PBS buffer containing 0.1% propidium iodide. The cells were analyzed by fluorescent flow cytometry (FACScan; Becton Dickinson), and cells stained with propidium iodide were excluded from the analysis. The data presented are the mean fluorescent signal for the 10 000 cells collected. Measurements were also made for cells exposed to APTD conjugate peptides after they were subjected to PSA-mediated cleavage. One hundred microliters of 400 µM APTD conjugate solutions in PBS buffer were incubated with 10.8 µL (concentration ) 1.84 mg/mL) of enzymatically active PSA solution (Calbiochem) for ∼4 h at 37 °C. Samples of each of the resulting peptide solutions were analyzed by HPLC to establish the completeness of the cleavage. Each of the cleaved APTD peptide solutions was then incubated in triplicate with Jurkat cells as described above. Confirmation of Intracellular Uptake into PC3M Prostate Cancer Cells. Ten micromolar solutions of r8-K(FITC)NH2 (1), r4-K(FITC)NH2 (4), and DGGDGGDGGDCCHSSKLQG-r8K(FITC)NH2 (19) were preincubated with active PSA solution (5.4 µL, c ) 1.84 mg/mL for ∼4 h at 37 °C). The incubated peptides and conjugates were then added to 30 000 PC3M cells/ well (200 µL total volume) for 15 min. Following incubation, the cells were washed, and the samples were visualized using fluorescence laser confocal microscopy (wavelength λ ) 484



Scheme 2. Synthesis of APTD Conjugatesa

a

(a) 0.1% TFA in deionized water, 14-24 h, 90-95%.

nm). The PC3M cells were also imaged using the IVIS fluorescent camera, and the photon flux (photons/s) was measured. Data Analysis. All data were analyzed statistically using the Student’s t-test to determine the significance of differences in cell uptake of the various peptide conjugates.

RESULTS APTD Conjugate Library Synthesis and Purification. A peptide with sequence CHSSKLQG-r8-K, incorporating both the r8 PTD and linking sequences separated by a single glycine spacer, was synthesized with a fluorescein isothiocyanate (FITC) fluorophore attached to a side chain of lysine at the carboxyterminus. Multiple potential r8 PTD-attenuating sequences of the form C(D)x, CD(GD)x, and CD(GGD)x (x ) 1, 2, or 3) were synthesized, and each was subsequently conjugated to the CHSSKLQG-r8-K(FITC)NH2 peptide through a disulfide bond of the cysteine residues (Scheme 2). Limited peptide solubility, aggregation, or both were observed for r8 PTD-attenuating sequences with more than four aspartate residues; therefore, the library was not expanded beyond those peptides listed in Table 1. TFA counterions were subsequently ion-exchanged for chloride ion after the disulfide coupling of the CHSSKLQGr8-K(FITC)NH2 and r8 PTD-attenuating sequences. Serum Stability Studies. Peptide conjugates 14-19 (Table 1) were investigated for their stability in serum (43, 44). After 4 h of incubation in serum at 37 °C, the decomposition of the peptide conjugates ranged from 1% to 5%. The most effectively attenuated APTD conjugate 19 (DGGDGGDGGDCCHSSKLQGr8-K(FITC)NH2) was found to be 97% intact after 4 h of incubation at 37 °C. PSA Cleaving Studies. Following incubation in PSAcontaining phosphate buffer solution, tested APTD conjugates demonstrated three distinct peaks on analytical HPLC, where one of these peaks corresponded to uncleaved APTD conjugate while the other two peaks corresponded to the expected cleavage

products. Peak assignment was accomplished by collection of the new peaks following cleavage and determination of their masses by mass spectrometry (MALDI). The MALDI data indicated that proteolysis took place between glutamine and glycine residues of the PSA substrate (HSSKLQ-G), as predicted, giving products such as NH2-DGGDGGDGGDCHSSKLQ (MW calculated 1764.76, found 1766.12) and G-r8K(FITC)NH2 (MW calculated 1841.12, found 1843.38) in the case of the APTD conjugate 19 cleaving study. The time required to cleave half of the substrate varied between the APTD conjugates but was generally in the range of 1.8-2 h. Kinetics of PSA proteolysis of the most effectively attenuated APTD (peptide conjugate 19) is shown in Figure 1. No detectable decomposition was observed after incubation of the APTD conjugates in phosphate buffer solution without PSA enzyme at 37 °C for 4 h. These data were in agreement with that previously published by other groups (3, 36, 41-42). Differential APTD Uptake prior to and following PSAMediated Cleavage. The APTD conjugates composed of an r8 PTD, a PSA-selective cleavage sequence (HSSKLQG), and an r8 PTD-attenuating sequence (CD(GGD)x), where x ) 2 or 3 (compounds 18 and 19, respectively) were incubated in PSAcontaining media for 4 h prior to the introduction of either Jurkat or PC3M cells for 5 or 15 min, respectively. Flow cytometry and fluorescence laser confocal microscopy were utilized to assess intracellular peptide uptake. Control experiments consisted of cell incubation in peptide-containing media that had not been exposed to PSA, as well as incubation in solutions containing fluorescently labeled control compounds such as octamer of arginine (r8) 1 (high uptake) and tetramer (r4) or pentamer (r5) of arginine (compounds 3 and 4, insignificant uptake). The results of these studies are summarized in Figure 2, panel A (Jurkat cells) and panel B (PC3M prostate cancer cells). APTD cleavage by PSA significantly enhanced uptake in both Jurkat cells and prostate cancer cells at all time points and concentrations (p < 0.05);


Goun et al.

Figure 3. (A) Uptake of various concentrations of APTD conjugates incorporating r8 PTD, a PSA enzyme-specific sequence, and one of a library of r8 PTD-attenuating sequences composed of two aspartates spaced with different numbers of glycine residues (DD, DGD, DGGD, DGGGD). Standard deviations are shown for each point; however, their magnitudes were, in many cases, less than 5% of the datapoint. (B) Bar graph demonstrating APTD conjugate uptakes at a single concentration (20 µM), extracted from data shown in panel A. This experiment demonstrates that the number of glycine spacing residues changed the effectiveness of r8 PTD transport attenuation by as much as 2-fold, with the order of decreasing effectiveness of 2 (greatest) > 3 > 0 > 1 (p < 0.05 for difference of attenuation achieved with a spacer of two glycine residues when compared to all other glycine spacer lengths). Figure 2. Comparison of cell uptake of APTD with and without prior PSA-mediated peptide cleavage: (A) Jurkat cells. Jurkat cells were incubated with each APTD conjugate solution for 5 min and washed twice. Uptake of each peptide was measured using flow cytometry. The level of cell uptake of the precleaved PSA-targeting APTD was between the levels of uptake of uncleaved APTD and r8 PTD. Standard deviations are shown for each point; however, their magnitudes were, in many cases, less than 5% of the datapoint. (B) PC3M cells. In triplicate, row A contains r8-K(FITC)NH2 (1) positive control, row B r4-K(FITC)NH2 (4) negative control, row C DGGDGGDGGDCCHSSKLQG-r8-K(FITC)NH2 (19), and row D DGGDGGDGGDCCHSSKLQG-r8-K(FITC)NH2 (19) preincubated with enzymatically active PSA enzyme. This image was obtained using an IVIS 50 imaging system (Xenogen Corp., Alameda, CA) with the level of fluorescence indicated by a color scale ranging from black (no fluorescence) to white (greatest measured fluorescence). The level of fluorescence reflects the level of uptake of the APTD or nonattenuated peptide transporters (r8 or r4). Uptake of the PSA targeting APTD takes place only after release of r8 PTD-attenuating sequence via PSA-specific substrate hydrolysis, as is demonstrated by fluorescence in row D but not in row C. Uptake of the cleaved APTD approaches but does not equal that of the nonattenuated r8 PTD (row A). Panel C presents bar representation of uptake of fluorescently labeled PSA-targeting peptide 19 in PC3M prostate cancer cells with and without precleavage by PSA. FITC ) fluorescein isothiocyanate; r8 ) arginine octamer; all other letters correspond to standard single-letter abbreviations for amino acids in the order from C- to N-terminus.

however, cell uptake of cleaved APTD remained significantly less than that of nonattenuated r8 PTD (p < 0.05).

Cell Partitioning and Attenuation/Spacing Experiments. The ability of various r8 PTD-attenuating sequences with different spacing to prevent the r8 PTD-containing peptide conjugates from entering cells was characterized by incubating Jurkat cells with different concentrations of APTD. The results of these experiments are presented in Figure 3A,B. All of the tested r8 PTD-attenuating sequences significantly decreased cell uptake of the r8 PTD transporter when compared to r8 PTD alone at concentrations of 3 µM and above (p < 0.01). The r8 PTDattenuating sequence that included aspartic acid residues spaced with two glycines demonstrated the greatest transport-attenuating effect. The number of glycine spacing residues changed the effectiveness of attenuation by as much as 2-fold between the most and the least efficient r8 PTD-attenuating sequence. At concentrations of 3-12 µM, the attenuation of transport activity achieved when aspartate residues were spaced with two glycine residues was significantly greater than the attenuation achieved when they were spaced by zero, one, or three residues (p < 0.05). Otherwise, spacing of aspartate residues with three glycine residues tended to provide greater transport attenuation than spacing with zero or one glycine residue; however, the differences in attenuation were not significant. The level of r8 PTD transport attenuation increased significantly with each aspartate residue added (p < 0.05) until three aspartate residues had been included. At most concentrations tested, the level of transport attenuation achieved by the


Figure 4. Characterization of the efficiency of r8 PTD transport attenuation of APTDs with increasing number of apartates (0-4) in comparison with the control compounds r8-K(FITC)NH2 (1) (high uptake) and r5-K(FITC)NH2 (3) (no significant uptake). Abbreviations: r8 ) arginine octamer; r5 ) arginine 5-mer; all other letters correspond to standard single-letter abbreviations for amino acids.

inclusion of four aspartate residues was not significantly different from that obtained with three. The presence of four aspartates in the r8 PTD-attenuating sequence exhibited a low level of uptake in Jurkat cells that was not significantly different from the level of uptake in r5 and r4 controls (p ) 0.08-0.36) (Figure 4). Results from the APTD 19, which demonstrated the best attenuation, were compared to r5 and r4 controls using PC3M cells. The results summarized in Figure 2B demonstrated similar inhibition of uptake between intact APTDs and control peptides (no significant difference).

DISCUSSION We report the development of a group of APTD conjugates that incorporate an r8 PTD connected to an r8 PTD-attenuating sequence via a protease cleavable linker ([r8 PTD-attenuating sequence]-[cleavable linker]-[r8 PTD]). The APTD conjugates themselves do not readily enter cells because the r8 PTDattenuating sequence disables the r8 PTD molecular transporter through internal complexation, but upon cleavage of the linker by a specific protease, the activity of the r8 PTD transporter is restored and cell entry of the cargo-carrying r8 PTD is observed. Although PTDs have been used by many investigators to facilitate the delivery of imaging agents and therapeutics in vitro and in vivo, the selective delivery of PTDs and their cargos to specific cell types is only beginning to be investigated. This initial research has mainly focused on antibody- and liposomemediated strategies (33, 45-47). Although antibodies have occasionally been successful in targeting tumors (48), their bulk hinders penetration of solid tumors and excretion of unbound reagent (49), and elaborate reengineering is required to minimize immunogenicity (50, 51). Other investigators have recently reported the development of molecules selectively taken up by cancers expressing matrix metalloproteinase-2 (MMP-2) or MMP-9 following cleavage of the molecule to release a labeled polyarginine transporting peptide (40). By incorporation of the selective transporter component into the small molecule, there is greater potential for molecular delivery into deep tissues. The present work provides further evidence for the utility of this promising selective targeting strategy for cargo delivery based on the level of local protease expression. In addition, the results presented here emphasize the achievements in decreased molecular size and specific targeting that can be accomplished by careful engineering of the molecular components of APTDs. In this study, a modular approach was taken during the design and synthesis of the APTDs, and many combinations of modules


containing (1) the r8 PTD and cleavable linker and (2) one of several r8 PTD-attenuating sequences were tested. The r8 PTD itself is a well-established facilitator of molecular transport across cell membranes. Although the detailed mechanisms and subcellular localization of these transporters is not fully understood and is expected to vary as a function of conjugate size, we have recently reported a mechanistic hypothesis for how water-soluble guanidinium-rich transporters attached to small cargoes (MW ca.

Intracellular Cargo Delivery by an Octaarginine Transporter Adapted

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