Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a

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Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a Murine Model of Prostate Cancer Wojciech G. Lesniak, Srikanth Boinapally, Sangeeta Ray Banerjee, Babak Behnam Azad, Catherine A. Foss, Chentian Shen, Ala Lisok, Bryan Wharram, Sridhar Nimmagadda, and Martin G Pomper Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00181 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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Molecular Pharmaceutics

Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a Murine Model of Prostate Cancer Wojciech G. Lesniak1, Srikanth Boinapally1, Sangeeta Ray Banerjee1, Babak Behnam Azad1, Catherine A. Foss1, Chentian Shen,1,2, Ala Lisok1, Bryan Wharram,1 Sridhar Nimmagadda1 and Martin G. Pomper1*

1Russell

H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, Maryland, 21287

2Department

of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China

*Correspondence to:

Martin G. Pomper, M.D., Ph.D. Johns Hopkins Medical Institutions 601 N Caroline St, Baltimore, MD 21287 Baltimore, MD 21287-910 Phone: 410-955- 2789 Fax: 443-817-0990 Email: [email protected].

No conflict of interest present.

Key Words: prostate cancer, prostate-specific membrane antigen, molecular imaging, poly(amidoamine), copper-64 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The prostate-specific membrane antigen (PSMA) is a validated target for detection and management of prostate cancer (PC). It has also been utilized for targeted drug delivery through antibody-drug conjugates and polymeric micelles. Polyamidoamine (PAMAM) dendrimers are emerging as a versatile platform in a number of biomedical applications due to their unique physicochemical properties including small size, large number of reactive terminal groups, bulky interior void volume and biocompatibility. Here we report the synthesis of generation-4 PSMAtargeted PAMAM dendrimers [G4(MP-KEU)] and evaluation of their targeting properties in vitro and in vivo using an experimental model of PC. A facile, one-pot synthesis gave nearly neutral nanoparticles with a narrow size distribution of 5 nm in diameter and a molecular weight of 27.3 kDa. They exhibited in vitro target specificity with a dissociation constant (Kd) of 0.32 ± 0.23 µM and preferential accumulation in PSMA+ PC3 PIP tumors vs. isogenic PSMA- PC3 flu tumors. Positron emission tomography-computed tomography (PET-CT) imaging and ex vivo biodistribution studies of dendrimers radiolabeled with 64Cu, [64Cu]G4(MP-KEU), demonstrated high accumulation in PSMA+ PC3 PIP tumors at 24 h post-injection (45.83 ± 20.09 percentage injected dose per gram of tissue, %ID/g) demonstrating a PSMA+ PC3 PIP/PSMA- PC3 flu ratio of 7.65 ± 3.35 at 24 h post-injection. Specific accumulation of G4(MP-KEU) and [64Cu]G4(MPKEU) in PSMA+ PC3 PIP tumors was inhibited by the known small-molecule PSMA inhibitor, ZJ-43. On the contrary, G4(Ctrl), control dendrimers without PSMA targeting moieties, showed comparable low accumulation of ~1 %ID/g in tumors irrespective of PSMA expression, further confirming PSMA+ tumor-specific uptake of G4(MP-KEU). These results suggest that G4(MPKEU) may represent a suitable scaffold by which to target PSMA-expressing tissues with imaging and therapeutic agents.

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INTRODUCTION Prostate-specific membrane antigen is overexpressed in the epithelium of most prostate cancers (PC), compared to normal prostate tissue and benign hyperplasia, and has been associated with castration-resistant PC, metastasis and poor prognosis.1-4 PSMA is also expressed on endothelial cells in the neovasculature of solid tumors other than PC, including lung, kidney, colon, stomach, breast and brain.5-9 The identification of PSMA substrate recognition sites has triggered extensive research leading to development of numerous lowmolecular-weight (LMW) PSMA inhibitors.10,11 Those agents have been radiolabeled with several different radioisotopes and used for detection of PSMA expression in a variety of cancers with positron emission tomography (PET) and single photon emission computed tomography (SPECT).10,11 Among LMW PSMA inhibitors, the most widely studied are the Lys-Glu-ureabased analogs, due to their facile synthesis, high PSMA binding affinity, specificity and rapid internalization.12-14 Some PSMA-targeted urea analogs are becoming important tools in the management of patients with prostate and other types of solid cancer, not only for detection and therapeutic monitoring, but also for endoradiotherapy.9-11,15-19 PSMA expression in solid cancers has also been successfully imaged with radiolabeled monoclonal antibodies, antigen-binding fragments (Fab2 and Fab’) and nanobodies in pre-clinical and clinical settings.20-22 In addition to numerous compounds for radionuclide imaging modalities, PSMA-specific agents for optical, magnetic resonance, photoacoustic and ultrasound imaging have been developed.23-28 PSMA has also been utilized for specific delivery of chemotherapeutics to solid tumors using antibody-drug conjugates (ADCs) and polylactic acid-polyethylene glycol (PLA-PEG)based polymeric nanoparticles (BIND-014), which have undergone clinical evaluation.29-31 Other PSMA-targeted nanoplatforms such as aptamers, bionized nanoferrite (BNF), lipid-nanocarrier, polyethyleneimine-plasmid polyplex (pDNA-PEI) and iron oxide magnetic nanoparticles have 3 ACS Paragon Plus Environment


been evaluated in pre-clinical studies.32-36 That array of platforms suggests the versatility of targeting PSMA in medicine. Thomas et al. demonstrated PSMA-mediated in vitro uptake of generation-5 PAMAM dendrimers conjugated with fluorescein and J591 anti-PSMA monoclonal antibody.37,38 In a follow-up study the same group showed specific in vitro toxicity for PAMAM dendrimer covalently modified with methotrexate and the Lys-Glu-urea PSMA targeting moiety.39 Those studies provided the rationale for development of PSMA-targeted PAMAM dendrimer-drug nanocarriers based on LMW targeting agents. However, data regarding in vivo specificity, biodistribution and clearance for PSMA-targeted dendrimers have not yet been reported. The advantage of small PAMAM nanoparticles (4 to 6 nm) compared to the aforementioned relatively large ADCs or polymeric nanoparticles (50 - 100 nm), is their low off-target tissue uptake and preferential active tumor accumulation mediated by the attached LMW targeting moieties.40 Our aim was to synthesize and evaluate the PSMA targeting properties of an optimized PAMAM dendrimer in vitro and in vivo. G4(MP-KEU) nanoparticles demonstrated in vitro specificity and preferential accumulation in PSMA+ rather than PSMA- isogenic xenografts in mice. In contrast, G4(Ctrl) control nanoparticles showed very low accumulation in both tumors.

EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma-Aldrich or Fisher Scientific unless otherwise specified. Ethylenediamine core amine-terminated generation-4 polyamidoamine dendrimer [G4(NH2)64] was acquired from Dendritech (Midland, MI). 64CuCl2 (t1/2 = 12.7 h) and 111InCl 3

(t1/2 = 2.805 days) were obtained from Washington University (St. Louis, MO) and


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Nordion (Ottawa, ON, Canada), respectively. 1,4,7,10-Tetraazacyclododecane-1,4,7,10tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) and N-[[[(1S)-1-carboxy3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ-43) were purchased from Macrocyclics (Plano, TX) and Tocris Bioscience (Minneapolis, MN), respectively. L-Glutamic acid di-tertbutyl ester hydrochloride and Nε-Z-L-lysine tert-butyl ester hydrochloride were purchased from Chem-Impex International, Inc (Wood Dale, IL). All reagents and solvents were used as received without further purification.

Synthesis of 5-mercaptopentanamido-Lys-Glu-urea (MP-KEU). The synthesis of thiol-terminated MP-KEU commenced with the transformation of commercially available bromovaleric acid into 5-(tritylthio)pentanoic acid by treating with triphenyl methanethiol in the presence of sodium methoxide according to a previously reported protocol.41 The trityl derivative was converted to (Ph)3MP-KEU upon reaction with previously reported Glu-Lys-urea42 in the presence of N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) and N,N-diisopropylethylamine (DIPEA). Subsequent removal of trityl and tertiary butyl groups was achieved using a TFA/H2O/ethanedithiol cocktail and product was purified by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) followed by lyophilization, which afforded the PSMA-targeting moiety MP-KEU as a white solid in 42 % overall yield.

5-(Tritylthio)pentanoic acid. To the oven-dried round bottom flask 150.8 mg (0.545 mmol, 1.0 eq) of trityl mercaptan was added and dissolved in 2 mL of dry toluene (obtained from a new, previously unopened bottle) with continuous stirring followed by the addition of a 30% (w/w) solution of sodium methoxide in methanol (220 μL, 1.2 mmol, 2.2 eq). To that mixture a 5 ACS Paragon Plus Environment


solution of bromovaleric acid (108.6 mg, 0.599 mmol, 1.1 eq) in methanol (1 mL) was slowly added at 5-10°C. The temperature of the reaction mixture was raised to 50°C, which then stirred for 2 h. The solvent was removed under reduced pressure and the residue was dissolved in 10 mL of water. The resulting aqueous solution was acidified (pH ~5-6) with 0.1 M H2SO4 and extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was recrystallized from EtOAc/hexanes to afford 5-(tritylthio)pentanoic acid as a white crystalline solid (142 mg, 70%). 1H-NMR

(500 MHz, CDCl3): δ 7.43-7.37 (m, 6H), 7.31-7.22 (m, 6H), 7.21-7.15 (m, 3H), 2.19 (t,

J = 2.2 Hz, 2H), 2.14 (t, J = 2.1 Hz, 2H), 1.60-1.51 (m, 2H), 1.44-1.35 (m, 2H); 13C-NMR (125 MHz, CDCl3): δ 179.8, 144.8, 129.6, 127.9, 126.6, 66.7, 33.5, 31.5, 28.1, 24.0.

Tri-tert-butyl(13S,17S)-7,15-dioxo-1,1,1-triphenyl-2-thia-8,14,16-triazanonadecane13,17,19-tricarboxylate. 5-(Tritylthio)pentanoic acid (120 mg, 0.319 mmol, 1.0 eq) and TSTU (96 mg, 0.319 mmol, 1.0 eq) were dissolved in 2 mL of DMF. Glu-Lys-urea (187 mg, 0.351 mmol, 1.1 eq) and DIPEA (144 mg, 1.11 mmol, 3.5 eq) dissolved in 2 mL of DMF were then added dropwise to the reaction mixture for 10 min. The resulting solution was stirred overnight at room temperature. The solvent was removed under reduced pressure and purified through silica gel chromatography using EtOAc/hexanes (50% EtOAc in hexanes) to afford (Ph)3MPKEU as a white solid (215 mg, 80%). 1H-NMR (500 MHz, CDCl3): δ 7.39 (d, J = 7.7 Hz, 5H), 7.32-7.17 (m, 8H), 7.20 (t, J = 7.4 Hz, 3H), 6.15-5.95 (m, 1H), 5.50-5.15 (m, 2H), 4.35-4.20 (m, 2H), 3.30-3.07 (m, 2H), 2.40-2.22 (m, 2H), 2.14 (t, J = 7.2 Hz, 2H), 2.10-2.02 (m, 3H), 1.90-1.68 (m, 2H), 1.63-1.53 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H), 1.43 (s, 9H), 1.52-1.27 (m, 6H); 13C-NMR (125 MHz, CDCl3): δ 173.2, 173.1, 172.4, 172.2, 157.3, 145.0, 129.6, 127.9, 126.6, 82.4, 81.6,


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80.6, 66.4, 53.5, 53.1, 39.1, 36.1, 32.5, 31.7, 29.0, 28.3, 28.1, 25.3, 22.9; MS (ESI): m/z 868.4 (M + Na).

(S)-1-Carboxy-5-(5-mercaptopentanamido)pentyl)carbamoyl)-L-glutamic acid (MPKEU). A 5 mL mixture of TFA/H2O/ethanedithiol (94:3:3) was added to the round bottom flask containing (Ph)3MP-KEU (100 mg, 0.118 mmol) at 0oC. The reaction mixture was stirred for 3 h at room temperature (RT) and concentrated under reduced pressure. The crude product was purified by preparative RP-HPLC chromatography using 0.1% TFA in H2O and 0.1% TFA in acetonitrile as eluents followed by lyophilization to afford compound MP-KEU as a white solid (38.5 mg, 75%). RP-HPLC purification was achieved using Agilent System, λ=220 nm, 250×10 mm Phenomenex Luna C18 column, solvent gradient: 90% H2O (0.1% TFA) and 10% ACN (0.1% TFA), reaching 60% of ACN in 20 min at a flow rate of 5 mL/min, product eluted at 8.7 min. 1H-NMR (400 MHz, CDCl3): δ 7.75 (t, J = 5.7 Hz, 1H), 6.30 (dd, J = 8.4, 13.0 Hz, 2H), 4.12-3.99 (m, 2H), 2.99 (q, J = 6.2 Hz, 2H), 2.52-2.47 (m, 1H), 2.44 (t, J = 6.6 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.1 Hz, 2H), 1.97-1.85 (m, 1H), 1.77-1.19 (m, 10H); MS (ESI): m/z 436.1 (M + H).

Synthesis of G4(MP-KEU) nanoparticles. Preparation of G4(MP-KEU) nanoparticles involved a multi-step synthesis as presented in Scheme 1B. In the first step G4(NH2)64 dendrimer (0.229 g, 1.61×10-5 mol) was dissolved in 10 mL of PBS buffer (0.1 M, pH = 7.4), placed in a round bottom flask and 2 mol equivalents of DOTA-NHS-ester (0.0245 g, 3.22×10-5 mol) reconstituted in 0.2 mL of DMSO was added. After 2 h of stirring at RT the reaction mixture was subjected to dialysis against deionized water using a regenerated cellulose membrane with a 7 ACS Paragon Plus Environment


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10,000 Da molecular weight cut-off (MWCO). Excess water was then evaporated and the residue was lyophilized, which provided 0.249 g of the G4(NH2)(DOTA) conjugate. Conjugation of rhodamine, MP-KEU and capping of primary amine with butane-1,2-diol was achieved in a onepot synthesis. Next, 0.0219 g (1.58×10-6 mol) of G4(NH2)(DOTA) conjugate was dissolved in 5 mL of PBS and mixed with 0.1 mL of DMSO containing 0.0038g (5.48×10-6 mol) of rhodamineNHS ester. After 2 h of stirring at RT an aliquot of the reaction mixture was subjected to MALDI-TOF mass spectrometry (as described below) to confirm conjugation of rhodamine with G4(NH2)(DOTA)

conjugate.

Next,

0.0078

g

(2.33×10-5

mol)

succinimidyl-4-[N-

maleimidomethyl]cyclohexane-1-carboxylate (SMCC) heterobifunctional linker dissolved in 0.1 mL of DMSO was added and reacted for 1 h, followed by MADLI-TOF mass spectrometric analysis to confirm covalent attachment of SMCC linker with G4(NH2)(DOTA)(rhodamine) conjugate. Subsequently, 0.0127 g (2.99×10-5 mol) of MP-KEU dissolved in 0.5 mL of PBS (c=0.1 M, pH=7.4) was added to the reaction mixture and was allowed to react for 1 h. Following that, MALDI-TOF mass spectrometry confirmed covalent attachment of the PSMAtargeting

moieties

to

the

G4(NH2)(DOTA)(rhodamine)(MCC)

maleimide-activated

nanoparticles. Then 0.1 mL of 4 M NaOH aqueous solution and 0.2 mL (2.99×10-3 mol) of glycidol was added and the reaction was continued for an additional 16 h to cap the remaining unmodified primary amines with butane-1,2-diol and provide G4(MR-KEU), PSMA-targeted dendrimers. G4(MP-KEU) was initially purified using a PD10-sized exclusion column (GE Healthcare), followed by purification on a RP-HPLC system (Varian ProStar) equipped with a 1260 Infinity photodiode array detector (Agilent) using a semi-preparative C-18 Luna column (5 mm, 10×25 mm Phenomenex) and gradient elution. The gradient proceeded as: 98% H2O (0.1% TFA) and 2% acetonitrile (0.1% TFA), reaching 100% of acetonitrile (0.1% TFA) in 30 min at a


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flow rate of 4 mL/min. G4(MP-KEU) was collected at between 10 and 13 min of elution. That fraction was evaporated using a rotary evaporator, and the obtained residue was dissolved in deionized water and lyophilized, yielding 0.031 g of red powder.

Synthesis of control G4(Ctrl) nanoparticles. G4(NH2)(DOTA) conjugate (Scheme 1) (0.0205 g, 1.47×10-6 mol) was dissolved in 5 mL of PBS and mixed with rhodamine-NHS ester (0.004 g, 5.77×10-6 mol in 0.1 mL of DMSO) and was allowed to react for 2 h at RT. MALDITOF analysis was performed on an aliquot of the reaction mixture, which was then followed by the addition of 4 M NaOH (0.1 mL, aqueous solution) and 0.2 mL (2.99×10-3 mol) of glycidol. The resulting reaction mixture was stirred overnight at RT to cap the remaining primary amines with butane-1,2-diol. G4(Ctrl) nanoparticles were purified using Amicon centrifugal filters with 10,000 Da MWCO (Millipore Sigma, Rockville MD) and deionized water, followed by lyophilization, which ultimately provided 0.025 mg of red material.

Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry. Spectra of G4(NH2)64, intermediate conjugates, final G4(MP-KEU) and G4(Ctrl) nanoparticles were recorded on a Voyager DE-STR spectrometer, using 2,5-dihydroxybenzoic acid (DHB) as a matrix. Matrix was dissolved in 50% MeOH and 0.1% TFA aqueous solution and conjugates obtained from reaction mixture were passed through zeba spin desalting columns with 7,000 Da MWCO (ThermoFisher Scientific, Waltham MA). Matrix (10 µL) at a concentration of 10 mg/mL was mixed with 10 µL of dendrimer at a concentration of 4 mg/mL. Next, 1 µL of the resulting mixture was placed on the target plate (in triplicate) and evaporated. The number of shots and laser power were adjusted according to spectrum quality.



Dynamic light scattering and zeta potential measurments. Dynamic light scattering (DLS) and zeta potential analyses were performed using a Malvern Zetasizer Nano ZEN3600. G4(MP-KEU) and G4(Ctrl) nanoparticles were prepared at a concentration of 2 mg/mL in PBS (c=0.1 M, pH 7.4). DLS measurements were performed at a 90° scattering angle at 25°C.

Cell lines and tumor models. PSMA+ PC3 PIP (high PSMA expression) and PSMAPC3 flu (low PSMA expression) cell lines were obtained from Dr. Warren Heston (Cleveland Clinic) and were maintained as previously described.43 Cells were grown to 80%-90% confluence before trypsinization and formulation in Hank’s balanced salt solution (HBSS, Sigma) for implantation into mice. Animal studies were performed according to protocols approved by the Johns Hopkins Animal Care and Use Committee (ACUC). For PET-CT imaging and ex vivo biodistribution studies, intact male NOD-SCID mice (Johns Hopkins University, inhouse colony) were implanted subcutaneously in the upper opposite flanks with 1×106 PSMA+ PC3 PIP and PSMA- PC3 flu cells behind either shoulder. Mice were used for imaging or ex vivo biodistribution studies when the tumor xenografts reached 3 to 5 mm in diameter.

In vitro binding assay. To evaluate specificity and affinity of G4(MP-KEU) to PSMA three different assays were carried out. (1) Approximately 1×106 PSMA+ PC3 PIP and PSMAPC3 flu cells were suspended in FACS buffer (PBS, 2 mM EDTA, 0.5% FBS), placed in FACS tubes, followed by addition of G4(MP-KEU) to achieve final concentrations ranging from 10 nM to 0.64 µM, followed by incubation with gentle rocking for 30 min at RT. Cells were then washed three times with 1 mL of cold PBS, transferred to a 96-well plate and fluorescence was read using a Perkin Elmer Victor3V 1420 multi-label counter with 550 nm excitation and 590 nm 10 ACS Paragon Plus Environment

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emission wavelengths. Cells incubated without G4(MP-KEU) were used to derive background fluorescence. To generate a PSMA-specific binding curve, fluorescence intensity detected in PSMA- PC3 flu cells was subtracted from signal detected in PSMA+ PC3 PIP cells. (2) Approximately 5×105 PSMA+ PC3 PIP cells were incubated with G4(MP-KEU) at concentrations ranging from 10 nM to 0.64 µM in the presence and absence of 1 mM of the known PSMA inhibitor, ZJ-43, and the same protocol as described above was applied. To generate a PSMA-specific binding curve, fluorescence intensity detected in PSMA+ PC3 PIP cells incubated with ZJ-43 and G4(MP-KEU) was subtracted from signal measured in PSMA+ PC3 PIP cells incubated with G4(MP-KEU) only. To derive Kd values, the PSMA-specific binding curves were fitted to a one-site binding hyperbola using GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA). (3) Approximately 7×105 PSMA+ PC3 PIP cells were incubated with G4(MP-KEU) at 1 µM with a serially concentrated solution of ZJ-43 (from 1 pM to 1 mM) for 30 min at RT, followed by washing and analysis on a plate reader as describe above. The IC50 value for ZJ-43 was calculated by fitting the data to a sigmoidal dose response curve using GraphPad Prism 4 software. All experiments were performed in triplicate.

Epifluorescence microscopy. To evaluate in vitro G4(MP-KEU) uptake in PSMA+ PC3 PIP and PSMA- PC3 flu cell lines, 1×106 cells/well (500 µL) were seeded in a chamber slide overnight. G4(MP-KEU) (5 µM in 15 µL) with or without 0.5 µL ZJ-43 (10 mM) was added to each well. After a 2 h incubation at 37oC, each well was washed twice in PBS for 5 min. After coverslip mounting the slide was viewed by using a Nikon 80i upright microscope (Nikon Instruments, Melville, NY) equipped with a Nikon DS-Qi1Mc darkfield CCD camera and excited by a Nikon Intensilight C-HGFI lamp. All images were recorded and processed using



Nikon Imaging software Elements (Nikon Instruments, Melville, NY). To assess accumulation of nanoparticles in PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and kidneys, tissues were harvested 24 h after intravenous injection of G4(MP-KEU) (75 µg) or G4(MP-KEU) (75 µg) and ZJ-43 (1 mg), were fresh frozen and cut at 20 µm thickness using a cryomicrotome. Sections were subsequently probed with mouse anti-human PSMA antibody (Abcam, GCP-05, 1:68) at RT for 1 h. After rinsing twice in PBS for 5 min, goat antimouse AlexaFluor® 680 (Invitrogen; 1:200) secondary antibody was added and incubated for 30 min at room temperature. Slides were then stained with Hoechst 33342 (Invitrogen; 1:1,000) for 1 min, rinsed twice in PBS for 5 min, and a cover slip was applied together with unstained sections from PSMA+ PC3 PIP, PSMA- PC3 flu xenografts and kidney. All slides were viewed and processed as described above.

Optical Imaging. Ex vivo biodistribution studies of G4(MP-KEU) and G4(Ctrl) nanoparticles were carried out in male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMAPC3 flu xenografts. Twenty-four hours after injection of G4(Ctrl) (64 µg, 2.76 nmol), G4(MPKEU) (75 µg, 2.75 nmol) or G4(MP-KEU) (75 µg, 2.75 nmol) plus ZJ-43 (25 mg/kg, 0.082 mol/kg, blocker) reconstituted in 200 µL of saline (n=4 for each group with dendrimer and n=2 for saline), the mice were sacrificed and selected organs and tumors were harvested and viewed on a Xenogen IVIS Spectrum optical imaging system (Caliper Life Sciences, Hanover, MD) with excitation at 535 nm and emission at 580 nm and a 3 s exposure. All images were collected and processed using Living Image Software v4.5. Organs and tumors dissected from mice injected with 200 µL of saline were used to determine background fluorescence. Subsequently, PSMA+ PC3 PIP and PSMA- PC3 flu tumors and kidneys were subjected to microscopic studies, as


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described above.

Radiolabeling. The radiolabeling of G4(MP-KEU) with 64Cu (t1/2 = 12.7 h) was carried out in 0.1 M aqueous solution of sodium acetate (pH 4.5) for 30 min at 85oC under conditions free of transition metals. Prior to incubation, 50 µg of G4(MP-KEU) (1.83 nmol) was mixed with 185 MBq of 64CuCl2 in 0.2 mL of sodium acetate buffer. To remove free and loosely bound 64Cu, ethylenediaminetetraacetic acid (EDTA) was added to the reaction mixture to achieve a final concentration of 5 mM. Following an additional 5 min of incubation, radio-HPLC analysis and purification with a Amicon Ultra centrifugal filters with 10,000 Da MWCO (Sigma-Aldrich, Milwaukee, USA) was performed. For further studies [64Cu]G4(MP-KEU) was diluted with saline. For radio-HPLC the same elution conditions as described for the G4(MP-KEU) purification was used. The same procedure was applied to prepare the [64Cu]G4(Ctrl) control radiotracer. The radiolabeling of G4(MP-KEU) and G4(Ctrl) with

111In

(t1/2 = 67.32 h) was carried out in a

0.2 M aqueous solution of ammonium acetate (pH 4.5) under conditions free of transition metals. G4(MP-KEU) (50 µg, 1.83 nmol) or G4(Ctrl) (2.15 nmol) was mixed with 185 MBq of 111InCl3 in 0.15 mL of labeling buffer and incubated for 60 min at 80oC, followed by ITLC with 0.1 M EDTA as the mobile phase to evaluate labeling efficiency. Purification was accomplished using Amicon Ultra centrifugal filters with 10,000 Da MWCO.

In vitro evaluation of

64Cu-

and

111In-labeled

G4(MP-KEU) and G4(Ctrl). In vitro

binding was carried out using 1×106 of PSMA+ PC3 PIP and PSMA- PC3 flu cells incubated with 37 KBq of the given radiotracer for 30 min at RT. After incubation, cells were washed three



times with cold PBS, followed by counting on an automated gamma counter (1282 Compugamma CS, Pharmacia/LKBNuclear, Inc., Gaithersburg, MD). To demonstrate PSMA specific binding, blocking was carried out with 1 mM of ZJ-43. All the in vitro uptake studies were performed in triplicate for each cell line and repeated three times. An internalization assay of [111In]G4(MP-KEU) or [111In]G4(Ctrl) was performed following our previous report.44

PET-CT imaging of mouse xenografts. Male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors were intravenously injected with approximately 8.51 MBq of [64Cu]G4(MP-KEU) in 200 L of saline (n = 3). Mice were initially anesthetized under 3% isoflurane in oxygen (2 L/min) and maintained at 1% isoflurane during scanning. For blocking experiments mice were co-injected with [64Cu]G4(MP-KEU) and 25 mg/kg of ZJ-43 in saline (n=1). PET images were acquired at the indicated times in two bed positions to obtain whole body images as 7 min per bed using an ARGUS small-animal PET/CT scanner (Sedecal, Madrid, Spain). A CT scan (512 projections) was performed at the end of each PET scan for anatomic coregistration. PET data were reconstructed using the two-dimensional ordered subsets-expectation maximization algorithm (2D-OSEM) and corrected for dead time and radioactive decay. Presented images were generated and visualized using Amira software (FEI, Hillsboro, OR).

SPECT-CT imaging and analysis. Whole-body SPECT-CT images of male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors were acquired on an X-SPECT small animal SPECT-CT system (Gamma Medica Ideas, Northridge, CA). After an intravenous injection of 29.6 MBq of [111In]G4(MP-KEU) or [111In]G4(Ctrl), mice were anesthetized under 3% isoflurane in oxygen (2 L/min) and maintained at 1% isoflurane during scanning (n=2).


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Images of two mice placed next to each other were acquired 5, 24 and 120 h post-injection. The tomographic data were acquired in 64 projections over 360o at 40 s per projection using medium energy pinhole collimators. CT was acquired in 512 projections to allow anatomic coregistration. Data were reconstructed using the ordered subsets-expectation maximization algorithm and 3D volume rendered images were generated using Amira 5.3.0 software (Visage Imaging, Inc.).

Ex vivo biodistribution of

64Cu-

and

111In-labeled

G4(MP-KEU) and G4(Ctrl).

Biodistribution studies were carried out in male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu xenografts using six groups injected intravenously with one of the following: 1.1 MBq of [64Cu]G4(MP-KEU) (group I, n=4); 1.1 MBq of [64Cu]G4(MP-KEU) plus 50 µg of G4(MP-KEU) (group II, n=3); 1.1 MBq of [64Cu]G4(MP-KEU) plus 1 mg of ZJ-43 (group III, n=3); 1.1 MBq of [64Cu]G4(Ctrl) (group 4, n=4); 1.1 MBq of [111In]G4(MP-KEU) containing approximately 10 µg of G4(MP-KEU) (group 5, n=4); or, 1.1 MBq of [111In]G4(Ctrl) containing approximately 10 µg of G4(Ctrl) (group 6, n=4). Mice were sacrificed at 3 h, 24 h and 48 h after administration of [64Cu]G4(MP-KEU) or [64Cu]G4(Ctrl), or at 1 h, 3 h, 24 h, 48 h, and 96 h after injection of [111In]G4(MP-KEU) or [111In]G4(Ctrl), and blood, tumors and selected organs were harvested and weighed. The radioactivity in collected samples was measured on a Perkin Elmer2480 Automatic Gamma Counter (PerkinElmer, Waltham, MA). To calculate the percent injected dose per gram of tissue (%ID/g), triplicate radioactive standards (10% of the injected dose) were counted along with tissue samples. Biodistribution data shown are mean ± the standard deviation of the mean.



Data analysis. An unpaired two-tailed t test using Prism 5 Software (GraphPad) was used for statistical analysis. P-values < 0.05 were considered to be significant.

RESULTS Synthesis and physicochemical characterization of G4(MP-KEU) and G4(Ctrl). Synthesis of the control and PSMA-targeted nanoparticles is presented in Scheme 1. Lys-Gluurea was modified with 5-mercaptopentanoic acid to form the MP-KEU (Scheme 1A) PSMA targeting moiety and facilitate its conjugation to dendrimer via reaction with maleimide of the SMCC heterobifunctional linker. MP-KEU was synthesized with high purity as demonstrated by RP-HPLC and ESI-Mass spectrometry (Figure 1A and B). G4(MP-KEU) was formulated by consecutive surface conjugation of DOTA, rhodamine, MCC and MP-KEU, followed by capping of the remaining terminal primary amines with butane-1,2-diol (Scheme 1B). G4(MP-KEU) nanoparticles were purified by RP-HPLC (Figure 1C), which yielded nanoparticles with a uniform RP-HPLC profile and UV-Vis spectrum, indicating covalent attachment of rhodamine with dendrimer (Figure 1D). To prepare G4(Ctrl) control nanoparticles, G4(NH2)62(DOTA)2 conjugate was reacted with rhodamine-NHS ester and glycidol (Scheme 1C). The average number of all conjugated moieties with dendrimers to formulate G4(MP-KEU) and G4(Ctrl) nanoparticles was derived from the consecutive increase of molecular weight upon each synthetic step as measured by MALDI-TOF (Figure 1E, Supporting Information Figure S1A). According to DLS analysis, the applied synthetic routes generated G4(MP-KEU) and G4(Ctrl) nanoparticles of similar narrow size distribution with a hydrodynamic radius of ~5 nm (Figure 1F, Supporting Information Figure S1B) and a zeta potential of -1.2 mV and -0.3 mV,


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respectively. The physicochemical properties of both G4(MP-KEU) and G4(Ctrl) nanoparticles targeted and control nanoparticles are collected in the Table 1.

In vitro evaluation of G4(MP-KEU) specificity. To assess G4(MP-KEU) specificity and affinity to PSMA in vitro, we carried out several different assays using isogenic human prostate cancer PSMA+ PC3 PIP and PSMA- PC3 flu cell lines (Figure 2). As presented in Figure 2A, a concentration-dependent increase of fluorescence intensity upon addition of G4(MP-KEU) was observed in PSMA+ PC3 PIP cells. In contrast, in PSMA- PC3 flu cells the signal intensity remained unchanged in the entire G4(MP-KEU) concentration range applied, suggesting specific binding of G4(MP-KEU) nanoparticles to PSMA with a derived Kd value of 0.49 µM (95% confidence interval 0.36 - 0.62 µM, Bmax = 1.91×106). Pre-mixing of PSMA+ PC3 PIP cells with 1 mM of ZJ-43 resulted in complete inhibition of G4(MP-KEU) uptake (Figure 2B) and provided a Kd value of 0.16 µM (95% confidence interval 0.10 - 0.22 µM, Bmax = 5.05×105). Next a competitive binding assay was carried out using PSMA+ PC3 PIP cells and 1 µM of G4(MPKEU) against varied concentration of ZJ-43 (Figure 2C). That assay provided an IC50 value of 1.22 µM (95% confidence interval 0.87 - 1.73 µM), indicating that a 10-fold higher concentration of ZJ-43 is required to inhibit interaction of G4(MP-KEU) with PSMA+ PC3 PIP cells. In vitro cellular uptake of G4(MP-KEU) by PSMA+ PC3 PIP and PSMA- PC3 flu cells was also evaluated by epifluorescence microscopy (Figure 2E). Internalization of G4(MP-KEU) was observed in PSMA+ PC3 PIP cells following 2 h of incubation at 37oC, which could be inhibited by excess ZJ-43. In contrast there was no detectable internalization of the G4(MP-



KEU) nanoparticles in PSMA- PC3 flu cells. Together, the above described findings demonstrate in vitro specificity and moderate affinity of G4(MP-KEU) for the target.

Optical imaging. Prompted by the promising in vitro results we undertook ex vivo evaluation of G4(MP-KEU) in NOD-SCID mice bearing subcutaneous PSMA+ PC3 PIP and PSMA- PC3 flu xenografts in opposite flanks with optical imaging. The upper panel of Figure 3 illustrates representative images of tissues dissected from mice 24 h after IV injection of G4(MPKEU) (A), G4(MP-KEU) plus ZJ-43 (B), G4(Ctrl) (C) and saline (D). High fluorescence intensity could be detected in PSMA+ PC3 PIP tumors obtained from mice treated with G4(MPKEU). Only marginally increased signal intensity compared to background was detected in PSMA- PC3 flu tumors, salivary glands, kidneys, pancreas, liver and bladder, indicating specific accumulation of G4(MP-KEU) in PSMA-expressing tumors. On the contrary, G4(Ctrl) nanoparticles did not show uptake in either PSMA positive or negative tumors and their presence could be detected only in kidneys and bladder (Figure 3C). Semi-quantitative analysis of G4(MP-KEU) accumulation in tumors provided a PSMA+ PC3 PIP/PSMA- PC3 flu ratio of 4.76 ± 0.02 (Figure 2E, F). Co-administration of G4(MP-KEU) with ZJ-43 resulted in decreased nanoparticle uptake in PSMA+ PC3 PIP tumors by more than 50% and complete clearance from kidneys, confirming PSMA-mediated uptake of G4(MP-KEU). To further evaluate accumulation of G4(MP-KEU), sections obtained from imaged PSMA+ PC3 PIP tumors, PSMA- PC3 flu tumors and kidneys were analyzed with epifluorescence microscopy (Figure 2G-N). In agreement with whole tumor and organ images, higher accumulation of G4(MP-KEU) within PSMA+ PC3 PIP tumors in comparison to PSMA- PC3 flu tumors and kidneys was detected in freshly cut, unstained sections (Figure 3G, H and I). After staining of PSMA and cell nuclei,


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fluorescence related to G4(MP-KEU) remained in samples obtained from PSMA+ PC3 PIP tumors (Figure 3J-N). The co-localization of PSMA expression and G4(MP-KEU) distribution further verified PSMA-mediated uptake of the nanoparticles in PSMA+ PC3 PIP tumors. In contrast, the same procedure resulted in failure to detect G4(MP-KEU) nanoparticles in samples acquired from PSMA- PC3 flu tumors and kidneys and PSMA+ PC3 PIP tumors obtained from blocking experiments (Supporting Information Figure S2).

Radiolabeling and in vitro evaluation. According to radio-HPLC, radiolabeling of G4(MP-KEU) with

64Cu

was achieved at 80.6% efficiency (Figure 4A). [64Cu]G4(MP-KEU)

was purified via centrifugal ultrafiltration, which yielded radiotracer with a specific activity of 70.67 MBq/nmol and 99.4 % radiochemical purity (Figure 4B). The same procedure yielded [64Cu]G4(Ctrl) control nanoparticles with a similar specific activity of 73.52 MBq/nmol and 99.5% radiochemical purity. [111In]G4(MP-KEU) and [111In]G4(Ctrl) were prepared with specific activity of 30.42 MBq/nmol and 77.32 MBq/nmol, respectively, and both were of radiochemical purity >99%. For evaluation in NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors [111In]G4(MP-KEU) and [111In]G4(Ctrl) were prepared with specific activity of 3 MBq/nmol by adding an appropriated amount of non-radiolabeled nanoparticles. Next, we evaluated in vitro uptake of [64Cu]G4(MP-KEU) and [64Cu]G4(Ctrl) in PSMA+ PC3 PIP and PSMS- PC3 flu cell lines. [64Cu]G4(MP-KEU) demonstrated higher uptake in PSMA+ PC3 PIP cells (Figure 4C, 12.92 ± 0.47 percent of incubated dose, %ID), compared to PSMA- PC3 flu (1.18 ± 0.58 %ID). The specific uptake of [64Cu]G4(MP-KEU) by PSMA+ PC3 PIP cells could be blocked with 1 mM of ZJ-43, further confirming the target specificity of radiolabeled targeted nanoparticles. ZJ-43 did not influence uptake of [64Cu]G4(MP-KEU) in



Page 20 of 50

PSMA- PC3 flu cells, which remained at 1.05 ± 0.25 %ID. In contrast, [64Cu]G4(Ctrl) showed similar low uptake of ~3 %ID in both PSMA+ PC3 PIP and PSMA- PC3 flu cell lines (data not shown). Similar in vitro uptake of 15.31 ± 0.97 %ID in PSMA+ PC3 PIP cells and 3.09 ± 0.59 %ID in PSMA- PC3 flu cells was observed for [111In]G4(MP-KEU). [111In]G4(Ctrl) showed 5.20 ± 0.39 and 3.47 ± 2.01 ID% in PSMA+ and PSMA- cells, respectively.

111In-G4(MP-KEU),

displayed > 80% internalization within 2 h in PSMA+ PC3 PIP cells and negligible internalization in PSMA- PC3 flu.

PET-CT imaging studies. To assess in vivo specificity of [64Cu]G4(MP-KEU), we performed PET-CT imaging of NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors in opposite flanks (n=5). In the preliminary studies, one mouse was injected with 7.4 MBq of [64Cu]G4(MP-KEU) and imaged at 1 h, 24 h and 48 h post-injection (Supporting Information Figure S3). PET-CT imaging acquired at 1 h after injection shows high background with the highest radioactivity accumulation in bladder and kidneys, followed by liver, spleen, lungs and heart, with modest PSMA+ PC3 PIP tumor uptake, which increased at later time points. To avoid intense signal in kidneys and bladder, further PET-CT imaging studies with [64Cu]G4(MP-KEU) started at 3 h after injection of [64Cu]G4(MP-KEU) (Figure 5A). Consistently, similar biodistribution of radioactivity was observed, except lower kidney accumulation, indicating fast renal clearance of [64Cu]G4(MP-KEU). Uptake of [64Cu]G4(MPKEU) in PSMA+ PC3 PIP tumors significantly increased by 24 h and remained high at 48 h after injection. Intravenous administration of [64Cu]G4(MP-KEU) resulted in high radioactivity uptake in liver and spleen, most likely due to trans-chelation of

64Cu

to endogenous proteins, such as

ceruloplasmin or albumin.45 Co-administration of [64Cu]G4(MP-KEU) with ZJ-43 led to


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inhibition of radioactivity accumulation in PSMA+ PC3 PIP tumors and to some extent in kidneys, in particular at 3 h post-injection, further demonstrating in vivo specificity of G4(MPKEU) nanoparticles.46,47

Ex vivo biodistribution of [64Cu]G4(MP-KEU) and [64Cu]G4(Ctrl). To validate the PET-CT imaging results, [64Cu]G4(MP-KEU) was further evaluated in ex vivo biodistribution studies using the same isogenic PSMA+ PC3 PIP and PSMA- PC3 flu tumor models (n=3 or 4). Figure 5B shows %ID/g of tissue in both tumors, blood and in selected organs in three different cohorts injected with [64Cu]G4(MP-KEU) (I), [64Cu]G4(MP-KEU) plus unlabeled G4(MP-KEU) (II) or [64Cu]G4(MP-KEU) plus ZJ-43 (III). Results obtained for cohort I showed consistently high accumulation of [64Cu]G4(MP-KEU) in PSMA+ PC3 PIP tumors with 30.56 ± 22.41 %ID/g at 3 h, 45.83 ± 20.09 %ID/g at 24 h and 26.11 ± 6.15 %ID/g at 48 h post-injection. In PSMAPC3 flu tumors %ID/g values were lower: 5.99 ± 0.58 %ID/g, 6.83 ± 1.00 %ID/g and 5.87 ± 0.94 %ID/g at the same time points, providing PSMA+/PSMA- tumor ratios of 4.22 ± 3.74, 7.65 ± 3.35 and 3.94 ± 1.09. Due to relatively long circulation of [64Cu]G4(MP-KEU) the PSMA+ PC3 PIP/blood ratio was 1.01 ± 0.90 at 3 h after injection, which increased to 6.68 ± 2.93 at 24 h and 5.81 ± 1.62 at 48 h after injection, when the blood pool concentration of [64Cu]G4(MP-KEU) decreased to 6.85 ± 0.85 %ID/g and 3.51 ± 0.8 %ID/g, respectively. PSMA+ PC3 PIP/muscle ratios were high at all time points. High accumulation of radioactivity was detected in kidneys and bladder at 3 h after injection, which significantly decreased at 24 h and 48 h after injection of [64Cu]G4(MP-KEU). In agreement with PET-CT imaging high retention of radioactivity was also detected in liver and spleen. Co-administration of [64Cu]G4(MP-KEU) with unlabeled G4(MP-KEU) resulted in decrease of radioactivity in all analyzed samples, particularly in blood,



Page 22 of 50

liver, spleen, kidneys, lacrimal glands and tumors. However, PSMA+ PC3 PIP/PSMA- PC3 flu, PSMA+ PC3 PIP/blood and PSMA+ PC3 PIP/muscle ratios remained comparable with those derived for mice injected with [64Cu]G4(MP-KEU) only, suggesting that the radiotracer maintained its PSMA specificity in spite of low specific activity. Co-administration of [64Cu]G4(MP-KEU) and ZJ-43 induced comparable effects to G4(MP-KEU), except considerably lower radioactivity was retained in PSMA+ PC3 PIP tumors. PSMA+ PC3 PIP/PSMA- PC3 flu ratios of 1 ± 0.46, 2.71 ± 0.79 and 1.88 ± 0.28 were demonstrated at 3 h, 24 h and 48 h after injection, further verifying PSMA-mediated accumulation of [64Cu]G4(MPKEU) in PSMA+ PC3 PIP xenografts. To further validate PSMA specificity of G4(MP-KEU), we assessed the ex vivo biodistribution [64Cu]G4(Ctrl) control dendrimers in the same tumor model (Figure

6).

[64Cu]G4(Ctrl) exhibited significantly faster clearance compared to [64Cu]G4(MP-KEU) and its blood concentration was only 0.34 ± 0.056 %ID/g at 3 h post-injection. Low radioactivity uptake, below 1 %ID/g, was detected in most analyzed tissues except kidneys (34.12 ± 7.58 %ID/g), liver (5.08 ± 0.82 %ID/g) and bladder (4.70 ± 3.94 %ID/g). Non-specific accumulation of [64Cu]G4(Ctrl) in PSMA+ PC3 PIP and PSMA- PC3 flu tumors was also low with 0.90 ± 0.06 and 0.71 ± 0.07 %ID/g, respectively, at 3 h post-injection. Biodistribution of [64Cu]G4(Ctrl) remained similar at 24 h and 48 h after injection, with slow clearance from kidneys.

Ex vivo biodistribution of [111In]G4(MP-KEU) and [111In]G4(Ctrl). To evaluate the potential effect of

64Cu2+

transchelation on biodistribution of [64Cu]G4(MP-KEU) and

[64Cu]G4(Ctrl) we radiolabeled both nanoparticles with thermodynamically stable complex with DOTA than does

111In3+,

64Cu2+,


which forms a more

and assessed their ex vivo

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biodistribution in NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors (Figure 7A). [111In]G4(MP-KEU) showed preferential uptake in PSMA+ PC3 PIP tumors compared to PSMA- PC3 flu tumors with 4.76 ± 1.34 %ID/g, 8.59 ± 2.47 %ID/g, 16.67 ± 3.63 %ID/g, 17.71 ± 3.81 %ID/g and 18.08 ± 1.55 %ID/g at 1, 3, 24, 48 and 96 h post-injection, and the PSMA+ PC3 PIP/PSMA- PC3 flu ratios of 2.38 ± 0.62, 3.1 ± 1.39, 4.03 ± 0.78, 4.66 ± 0.88 and 4.89 ± 1.68 at the same time points. [111In]G4(MP-KEU) exhibited relatively long circulation with 30.83 ± 2.71 %ID/g, 20.73 ± 4.16 %ID/g, 8.54 ± 0.66 %ID/g, 5.94 ± 1.09 %ID/g and 2.03 ± 0.15 %ID/g at 1, 3, 24, 48 and 96 h post-injection, respectively. A gradual decrease of radioactivity was observed within the heart (from 7.26 ± 1.17 %ID/g to 1.89 ± 0.11 %ID/g) and lungs (from 16.24 ± 2.24 %ID/g to 2.3 ± 0.1 %ID/g). Liver uptake of [111In]G4(MP-KEU) remained similar at all time points analyzed and ranged between 10.76 ± 0.94 %ID/g and 15.32 ± 2.82 %ID/g. Relatively low accumulation of [111In]G4(MP-KEU) in spleen was detected at 1 h (6.40 ± 0.8 %ID/g) and 3 h (7.63 ±1.91 %ID/g ) post-injection, which increased to 21.8 ± 4.9 %ID/g, 34.74 ± 11.14 %ID/g and 36.05 ± 6.04 %ID/g at 24 h, 48 h and 96 h post-injection, respectively. Accumulation of radioactivity in kidneys was 12.30 ±1.16 %ID/g at 1 h post-injection, decreasing to 4.98±0.48 %ID/g at 96 h. In contrast, [111In]G4(Ctrl) showed very low accumulation in tumors regardless of PSMA expression. Similarly to 64Cu radiolabeled G4(Ctrl), [111In]G4(Ctrl) rapidly cleared from blood stream with 0.46 ± 0.1 %ID/g at 1 h post-injection and major accumulation in kidneys (the highest being 71.33 ± 9.34 %ID/g at 3 h post-injection, which decreased to 21.13 ± 5.35 %ID/g at 96 h), followed by liver, reaching ~8 %ID/g at all time points analyzed.



SPECT-CT imaging of [111In]G4(MP-KEU) and [111In]G4(Ctrl) In agreement with ex vivo biodistribution analysis, SPECT-CT imaging (Figure 7B) confirmed preferential uptake of [111In]G4(MP-KEU) in PSMA+ PC3 PIP tumors vs. PSMAPC3 flu tumors. [111In]G4(MP-KEU) cleared from circulation at 120 h post-injection and could be detected only in PSMA+ PC3 PIP tumor, spleen and liver. Accumulation of [111In]G4(Ctrl) could be detected only in kidneys, bladder and liver with much lower retention of radioactivity compared to [111In]G4(MP-KEU) at 120 h.

4. DISCUSSION The aim of this study was to synthesize a PSMA-targeted PAMAM dendrimer and evaluate its in vitro and in vivo specificity. As a targeting moiety we have used the Lys-Glu-urea LMW inhibitor, as it was initially reported by our group to have suitable pharmacokinetics for in vivo targeting and imaging of PSMA and it has been conjugated with different nanoplatforms enabling PSMA-specific uptake.31,33,34 The biological properties of PAMAM dendrimers, such as pharmacokinetics, passive and active tumor targeting, strongly depend on their size and the nature of the terminal groups, which determine the net surface charge and hydrophobicity.48 Lower generation (up to generation 4, size ≤ 4 nm) PAMAM dendrimers form flexible scaffolds, which are rapidly excreted by the kidneys with minimal extravasation.48-50 Starting from generation 5 (size ≥ 5 nm), dendrimers form more rigid and globular nanoparticles that exhibit prolonged circulation with involvement of hepatobiliary excretion.40 Based on the above considerations, we hypothesized that application of generation 4 PAMAM dendrimers for synthesis of PSMA-targeted nanoparticles would promote their active targeting with major renal clearance. Indeed, our G4(MP-KEU) dendrimer conjugated with two DOTA chelators, three


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rhodamine dyes, 10 Lys-Glu-urea PSMA inhibitors and 12 hydrolyzed SMCC linkers, with most of the terminal amines capped with a butane-1,2-diol, showed preferential uptake in PSMAexpressing tumors, low retention in peripheral organs and preferential renal excretion. In comparison with LMW PSMA inhibitors, [64Cu]G4(MP-KEU) exhibited a relatively long circulation time with blood half-life greater than 6.2 h, which could be significantly reduced by co-injection with 50 µg of non-radiolabeled G4(MP-KEU) nanoparticles or 1 mg of the known PSMA inhibitor, ZJ-43. In contrast, G4(Ctrl) demonstrated a surprisingly 6-fold lower blood half-life (0.7-1.1 h), suggesting that conjugation of PSMA targeting moieties to the dendrimer is responsible for the extended residence time of G4(MP-KEU) in circulation. In agreement with PSMA expression in normal tissues, specific uptake of [64Cu]G4(MP-KEU) was detected in kidneys and lacrimal glands, as confirmed by blocking with ZJ-43 and unlabeled G4(MPKEU).46,51 Interestingly, inhibition of [64Cu]G4(MP-KEU) accumulation in PSMA+ PC3 PIP tumors by ZJ-43 was effective in decreasing radioactivity levels at 3 h after injection, comparable to the situation that obtains when attempting blockade of PSMA- PC3 flu tumors. However, at 24 h and 48 h, radiotracer uptake increased by ~50% in PSMA+ PC3 PIP tumors compared to PSMA- PC3 flu xenografts, most likely due to PSMA recycling52 and the availability of [64Cu]G4(MP-KEU) in circulation. Altogether, PET-CT imaging and ex vivo biodistribution studies demonstrate the high, specific uptake and retention of [64Cu]G4(MPKEU) in PSMA+ PC3 PIP tumors with consistently high PSMA+ PC3 PIP/PSMA- PC3 flu ratios, similar to the ratios obtained based on fluorescence detection. PSMA specificity of [64Cu]G4(MP-KEU) was further validated by marginal uptake of [64Cu]G4(Ctrl) in both PSMA+ and PSMA- tumors.



There was an apparent discrepancy in biodistribution of G4(MP-KEU) detected by optical imaging vs. its

64Cu-labeled,

radioactive counterpart, [64Cu]G4(MP-KEU), observed by

PET-CT imaging and ex vivo biodistribution. That manifested as high uptake of radioactivity within the liver and spleen on the PET-CT images, which we now attribute to the transchelation of 64Cu to endogenous protein such as ceruloplasmin.45 Indeed [111In]G4(MP-KEU), which will not promote such transchelation, showed considerably lower accumulation in liver and spleen, and similar uptake in all other tissues analyzed. Interestingly, [111In]G4(MP-KEU) displayed increased time-dependent splenic uptake, most likely due to opsonization and sequestration by the reticuloendothelial system, which might be reduced by PEGylation of this dendrimer. G4(MP-KEU) was designed with the structure and function of an antibody-drug conjugate (ADC) in mind. Reported therapeutically effective ADCs carry 3-4 drug molecules per antibody.53,54 Accordingly, G4(MP-KEU) was conjugated with 3-4 rhodamine molecules, which could be substituted with potent anticancer agents, most likely without affecting pharmacokinetics. If the therapeutically effective dose is ~6 mg/kg for an ADC,54 which is ~22 µmol/kg, then a similar degree of drug delivery would be possible with only ~4 µmol/kg of G4(MP-KEU) construct [since the MW of the ADC is about 5.5 times higher than that of G4(MP-KEU)]. We also have the option of increasing the number of conjugated drug molecules by utilizing additional terminal primary amines on the dendrimer, as needed. Additionally, [111In]G4(MP-KEU) displayed > 80% internalization within 2 h in PSMA+ PC3 PIP cells, which is comparable to our recently reported 5D3 anti-PSMA-antibody.55 However, the blood half-life of [111In]G4(MP-KEU) is shorter compared to 29 h observed for [111In]DOTA-5D3, so anticipated myelotoxicity would be lower for the dendrimer-drug conjugate. The G4(MP-KEU)


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also displayed much lower liver uptake compared to relatively large PSMA-targeted nanoparticles.31,33

5. CONCLUSIONS G4(MP-KEU) proved capable of specific, PSMA-targeted delivery in vivo. Favorable pharmacokinetics and capacity for structural manipulation make G4(MP-KEU) a flexible scaffold for delivery of PSMA-targeted imaging and therapeutic agents.

ACKNOWLEDGMENTS: This work was funded by CA134675, CA184228, CA183031, EB024495, and the Commonwealth Foundation.

SUPPORTING INFORMATION: MALDI-TOF spectra showing increase of the molecular weight upon each synthetic step leading to formation of G4(Ctrl), DLS of G4(Ctrl), Epifluorescence microscopy of PSMA+ PC3 PIP tumor section obtained from mouse injected with G4(MP-KEU) (75 µg) plus ZJ-43 (25 mg/kg) and PSMA- PC3 flu tumor section dissected from mouse injected with G4(MP-KEU), Decay corrected volume rendered PET-CT images of a mouse bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors injected with 7.4 MBq of [64Cu]G4(MP-KEU).



Page 28 of 50

REFERENCES 1.

Ghosh, A.; Heston, W. D. W. Tumor target prostate specific membrane antigen (PSMA)

and its regulation in prostate cancer. J Cell Biochem 2004, 91, (3), 528-539. 2.

Chang, S. S.; Reuter, V. E.; Heston, W. D. W.; Gaudin, P. B. Comparison of anti-

prostate-specific membrane antigen antibodies and other immunomarkers in metastatic prostate carcinoma. Urology 2001, 57, (6), 1179-1183. 3.

Wright, G. L.; Grob, B. M.; Haley, C.; Grossman, K.; Newhall, K.; Petrylak, D.; Troyer,

J.; Konchuba, A.; Schellhammer, P. F.; Moriarty, R.

Upregulation of prostate-specific

membrane antigen after androgen-deprivation therapy. Urology 1996, 48, (2), 326-334. 4.

Perner, S.; Hofer, M. D.; Kim, R.; Shah, R. B.; Li, H.; Moller, P.; Hautmann, R. E.;

Gschwend, J. E.; Kuefer, R.; Rubin, M. A. Prostate-specific membrane antigen expression as a predictor of prostate cancer progression. Human pathology 2007, 38, (5), 696-701. 5.

Mhawech-Fauceglia, P.; Zhang, S.; Terracciano, L.; Sauter, G.; Chadhuri, A.; Herrmann,

F. R.; Penetrante, R. Prostate-specific membrane antigen (PSMA) protein expression in normal and neoplastic tissues and its sensitivity and specificity in prostate adenocarcinoma: an immunohistochemical study using mutiple tumour tissue microarray technique. Histopathology 2007, 50, (4), 472-483. 6.

Wang, H. L.; Wang, S. S.; Song, W. H.; Pan, Y.; Yu, H. P.; Si, T. G.; Liu, Y.; Cui, X. N.;

Guo, Z. Expression of prostate-specific membrane antigen in lung cancer cells and tumor neovasculature endothelial cells and its clinical significance. Plos One 2015, 10, (5). 7.

Haffner, M. C.; Kronberger, I. E.; Ross, J. S.; Sheehan, C. E.; Zitt, M.; Muhlmann, G.;

Ofner, D.; Zelger, B.; Ensinger, C.; Yang, X. M. J.; Geley, S.; Margreiter, R.; Bander, N. H.


Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Prostate-specific membrane antigen expression in the neovasculature of gastric and colorectal cancers. Human pathology 2009, 40, (12), 1754-1761. 8.

Baccala, A.; Sercia, L.; Li, J. B.; Heston, W.; Zhou, M. Expression of prostate-specific

membrane antigen in tumor-associated neovasculature of renal neoplasms. Urology 2007, 70, (2), 385-390. 9.

Fragomeni, R. A. S.; Menke, J. R.; Holdhoff, M.; Ferrigno, C.; Laterra, J. J.; Solnes, L.

B.; Javadi, M. S.; Szabo, Z.; Pomper, M. G.; Rowe, S. P. Prostate-Specific Membrane AntigenTargeted Imaging With [F-18] DCFPyL in High-Grade Gliomas. Clin Nucl Med 2017, 42, (10), E433-E435. 10.

Kiess, A. P.; Banerjee, S. R.; Mease, R. C.; Rowe, S. P.; Rao, A.; Foss, C. A.; Chen, Y.;

Yang, X.; Cho, S. Y.; Nimmagadda, S.; Pomper, M. G. Prostate-specific membrane antigen as a target for cancer imaging and therapy. Q J Nucl Med Mol Im 2015, 59, (3), 241-268. 11.

Maurer, T.; Eiber, M.; Schwaiger, M.; Gschwend, J. E. Current use of PSMA - PET in

prostate cancer management. Nat Rev Urol 2016, 13, (4), 226-235. 12.

Zhou, J.; Neale, J. H.; Pomper, M. G.; Kozikowski, A. P. NAAG peptidase inhibitors

and their potential for diagnosis and therapy. Nat Rev Drug Discov 2005, 4, (12), 1015-1026. 13.

Kozikowski, A. P.; Nan, F.; Conti, P.; Zhang, J. H.; Ramadan, E.; Bzdega, T.;

Wroblewska, B.; Neale, J. H.; Pshenichkin, S.; Wroblewski, J. T. Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase). Journal of medicinal chemistry 2001, 44, (3), 298-301. 14.

Pomper, M. G.; Musachio, J. L.; Zhang, J. Z.; Scheffel, U.; Zhou, Y.; Hilton, J.; Maini,

A.; Dannals, R. F.; Wong, D. F.; Kozikowski, A. P. C-11-MCG: Synthesis, Uptake Selectivity,



and Primate PET of a Probe for Glutamate Carboxypeptidase II (NAALADase). Mol Imaging 2002, 1, (2), 96-101. 15.

Burger, I.; Kranzbuehler, B.; Nagel, H.; Huellner, M.; Stolzmann, P.; Eberli, D. PET/MR

further improves detection of Ga-68-PSMA imaging for early biochemical recurrence in prostate cancer. J Nucl Med 2017, 58. 16.

Rowe, S. P.; Gorin, M. A.; Hammers, H. J.; Javadi, M. S.; Hawasli, H.; Szabo, Z.; Cho,

S. Y.; Pomper, M. G.; Allaf, M. E. Imaging of metastatic clear cell renal cell carcinoma with PSMA-targeted F-18-DCFPyL PET/CT. Ann Nucl Med 2015, 29, (10), 877-882. 17.

Sheikhbahaei, S.; Afshar-Oromieh, A.; Eiber, M.; Solnes, L. B.; Javadi, M. S.; Ross, A.

E.; Pienta, K. J.; Allaf, M. E.; Haberkorn, U.; Pomper, M. G.; Gorin, M. A.; Rowe, S. P. Pearls and pitfalls in clinical interpretation of prostate-specific membrane antigen (PSMA)-targeted PET imaging. Eur J Nucl Med Mol I 2017, 44, (12), 2117-2136. 18.

Delker, A.; Fendler, W. P.; Kratochwil, C.; Brunegraf, A.; Gosewisch, A.; Gildehaus, F.

J.; Tritschler, S.; Stief, C. G.; Kopka, K.; Haberkorn, U.; Bartenstein, P.; Boning, G. Dosimetry for Lu-177-DKFZ-PSMA-617: a new radiopharmaceutical for the treatment of metastatic prostate cancer. Eur J Nucl Med Mol I 2016, 43, (1), 42-51. 19.

Rahbar, K.; Ahmadzadehfar, H.; Kratochwil, C.; Haberkorn, U.; Schafers, M.; Essler, M.;

Baum, R. P.; Kulkarni, H. R.; Schmidt, M.; Drzezga, A.; Bartenstein, P.; Pfestroff, A.; Luster, M.; Lutzen, U.; Marx, M.; Prasad, V.; Brenner, W.; Heinzel, A.; Mottaghy, F. M.; Ruf, J.; Meyer, P. T.; Heuschkel, M.; Eveslage, M.; Bogemann, M.; Fendler, W. P.; Krause, B. J. German Multicenter Study Investigating Lu-177-PSMA-617 Radioligand Therapy in Advanced Prostate Cancer Patients. J Nucl Med 2017, 58, (1), 85-90.


Page 30 of 50

Page 31 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20.

Elgamal, A. A. A.; Troychak, M. J.; Murphy, G. P. ProstaScint (R) scan may enhance

identification of prostate cancer recurrences after prostatectomy, radiation, or hormone therapy: Analysis of 136 scans of 100 patients. Prostate 1998, 37, (4), 261-269. 21.

Pandit-Taskar, N.; O'Donoghue, J. A.; Divgi, C. R.; Wills, E. A.; Schwartz, L.; Gonen,

M.; Smith-Jones, P.; Bander, N. H.; Scher, H. I.; Larson, S. M.; Morris, M. J. Indium 111labeled J591 anti-PSMA antibody for vascular targeted imaging in progressive solid tumors. Ejnmmi Res 2015, 5. 22.

Chatalic, K. L. S.; Veldhoven-Zweistra, J.; Bolkestein, M.; Hoeben, S.; Koning, G. A.;

Boerman, O. C.; de Jong, M.; van Weerden, W. M. A Novel In-111-Labeled Anti-ProstateSpecific Membrane Antigen Nanobody for Targeted SPECT/CT Imaging of Prostate Cancer. J Nucl Med 2015, 56, (7), 1094-1099. 23.

Chen, Y.; Dhara, S.; Banerjee, S. R.; Byun, Y.; Pullambhatla, M.; Mease, R. C.; Pomper,

M. G. A low molecular weight PSMA-based fluorescent imaging agent for cancer. Biochem Bioph Res Co 2009, 390, (3), 624-629. 24.

Chen, Y.; Chatterjee, S.; Lisok, A.; Minn, I.; Pullambhatla, M.; Wharram, B.; Wang, Y.

C.; Jin, J. F.; Bhujwalla, Z. M.; Nimmagadda, S.; Mease, R. C.; Pomper, M. G. A PSMAtargeted theranostic agent for photodynamic therapy. J Photoch Photobio B 2017, 167, 111-116. 25.

Ray, S.; Song, X. O. L.; Yang, X.; Minn, I. L.; Lisok, A.; Chatterjee, S.; Chen, J.; van

Zijl, P.; McMahon, M.; Pomper, M.

Salicylic Acid-Based Polymeric Agents for Targeted

Molecular Magnetic Resonance Imaging (MRI) of Prostate Cancer. J Nucl Med 2017, 58. 26.

Liu, G. S.; Banerjee, S. R.; Yang, X.; Yadav, N.; Lisok, A.; Jablonska, A.; Xu, J. D.; Li,

Y. G.; Pomper, M. G.; van Zijl, P. A dextran-based probe for the targeted magnetic resonance



imaging of tumours expressing prostate-specific membrane antigen. Nat Biomed Eng 2017, 1, (12), 977-+. 27.

Tavakoli, B.; Chen, Y.; Guo, X. Y.; Kang, H. J.; Pomper, M.; Boctor, E. M.

Multispectral photoacoustic decomposition with localized regularization for detecting targeted contrast agent. Proc Spie 2015, 9323. 28.

Wang, L. F.; Li, L.; Guo, Y. L.; Tong, H. P.; Fan, X. Z.; Ding, J.; Huang, H. Y.

Construction and In Vitro/In Vivo Targeting of PSMA-Targeted Nanoscale Microbubbles in Prostate Cancer. Prostate 2013, 73, (11), 1147-1158. 29.

Petrylak, D. P.; Smith, D. C.; Appleman, L. J.; Fleming, M. T.; Hussain, A.; Dreicer, R.;

Sartor, A. O.; Shore, N. D.; Vogelzang, N. J.; Youssoufian, H.; DiPippo, V. A.; Stambler, N.; Huang, K.; Israel, R. J. A phase 2 trial of prostate-specific membrane antigen antibody drug conjugate (PSMA ADC) in taxane-refractory metastatic castration-resistant prostate cancer (mCRPC). J Clin Oncol 2014, 32, (15). 30.

Galsky, M. D.; Eisenberger, M.; Moore-Cooper, S.; Kelly, W. K.; Slovin, S. F.; Delacruz,

A.; Lee, Y.; Webb, I. J.; Scher, H. I. Phase I trial of the prostate-specific membrane antigen directed immunoconjugate MLN2704 in patients with progressive metastatic castration-resistant prostate cancer. J Clin Oncol 2008, 26, (13), 2147-2154. 31.

Hrkach, J.; Von Hoff, D.; Ali, M. M.; Andrianova, E.; Auer, J.; Campbell, T.; De Witt,

D.; Figa, M.; Figueiredo, M.; Horhota, A.; Low, S.; McDonnell, K.; Peeke, E.; Retnarajan, B.; Sabnis, A.; Schnipper, E.; Song, J. J.; Song, Y. H.; Summa, J.; Tompsett, D.; Troiano, G.; Hoven, T. V.; Wright, J.; LoRusso, P.; Kantoff, P. W.; Bander, N. H.; Sweeney, C.; Farokhzad, O. C.; Langer, R.; Zale, S. Preclinical Development and Clinical Translation of a PSMA-


Page 32 of 50

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Targeted Docetaxel Nanoparticle with a Differentiated Pharmacological Profile. Sci Transl Med 2012, 4, (128). 32.

Farokhzad, O. C.; Cheng, J. J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J.

P.; Langer, R. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. P Natl Acad Sci USA 2006, 103, (16), 6315-6320. 33.

Azad, B. B.; Banerjee, S. R.; Pullambhatla, M.; Lacerda, S.; Foss, C. A.; Wang, Y. C.;

Ivkov, R.; Pomper, M. G.

Evaluation of a PSMA-targeted BNF nanoparticle construct.

Nanoscale 2015, 7, (10), 4432-4442. 34.

Zhu, C.; Bandekar, A.; Sempkowski, M.; Banerjee, S. R.; Pomper, M. G.; Bruchertseifer,

F.; Morgenstern, A.; Sofou, S.

Nanoconjugation of PSMA-Targeting Ligands Enhances

Perinuclear Localization and Improves Efficacy of Delivered Alpha-Particle Emitters against Tumor Endothelial Analogues. Molecular cancer therapeutics 2016, 15, (1), 106-113. 35.

Bhatnagar, A.; Wang, Y.; Mease, R. C.; Gabrielson, M.; Sysa, P.; Minn, I.; Green, G.;

Simmons, B.; Gabrielson, K.; Sarkar, S.; Fisher, P. B.; Pomper, M. G. AEG-1 promotermediated imaging of prostate cancer. Cancer research 2014, 74, (20), 5772-81. 36.

Tse, B. W. C.; Cowin, G. J.; Soekmadji, C.; Jovanovic, L.; Vasireddy, R. S.; Ling, M. T.;

Khatri, A.; Liu, T. Q.; Thierry, B.; Russell, P. J.

PSMA-targeting iron oxide magnetic

nanoparticles enhance MRI of preclinical prostate cancer. Nanomedicine-Uk 2015, 10, (3), 375386. 37. R., Jr.

Thomas, T. P.; Patri, A. K.; Myc, A.; Myaing, M. T.; Ye, J. Y.; Norris, T. B.; Baker, J. In vitro targeting of synthesized antibody-conjugated dendrimer nanoparticles.

Biomacromolecules 2004, 5, (6), 2269-74.



38.

Page 34 of 50

Patri, A. K.; Myc, A.; Beals, J.; Thomas, T. P.; Bander, N. H.; Baker, J. R. Synthesis and

in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem 2004, 15, (6), 1174-1181. 39.

Huang, B. H.; Otis, J.; Joice, M.; Kotlyar, A.; Thomas, T. P. PSMA-Targeted Stably

Linked "Dendrimer-Glutamate Urea-Methotrexate" as a Prostate Cancer Therapeutic. Biomacromolecules 2014, 15, (3), 915-923. 40.

Sadekar, S.; Ray, A.; Janat-Amsbury, M.; Peterson, C. M.; Ghandehari, H. Comparative

Biodistribution of PAMAM Dendrimers and HPMA Copolymers in Ovarian-Tumor-Bearing Mice. Biomacromolecules 2011, 12, (1), 88-96. 41.

Majer, P.; Jackson, P. F.; Delahanty, G.; Grella, B. S.; Ko, Y. S.; Li, W.; Liu, Q.; Maclin,

K. M.; Polakova, J.; Shaffer, K. A.; Stoermer, D.; Vitharana, D.; Wang, E. Y.; Zakrzewski, A.; Rojas, C.; Slusher, B. S.; Wozniak, K. M.; Burak, E.; Limsakun, T.; Tsukamoto, T. Synthesis and biological evaluation of thiol-based inhibitors of glutamate carboxypeptidase II: discovery of an orally active GCP II inhibitor. Journal of medicinal chemistry 2003, 46, (10), 1989-96. 42.

Maresca, K. P.; Hillier, S. M.; Femia, F. J.; Keith, D.; Barone, C.; Joyal, J. L.;

Zimmerman, C. N.; Kozikowski, A. P.; Barrett, J. A.; Eckelman, W. C.; Babich, J. W. A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer. Journal of medicinal chemistry 2009, 52, (2), 347-57. 43.

Mease, R. C.; Dusich, C. L.; Foss, C. A.; Ravert, H. T.; Dannals, R. F.; Seidel, J.;

Prideaux, A.; Fox, J. J.; Sgouros, G.; Kozikowski, A. P.; Pomper, M. G.

N-[N-[(S)-1,3-

dicarboxypropyl]carbamoyl]-4-[F-18]fluorobenzyl-L-cysteine, [F-18]DCFBC: A new imaging probe for prostate cancer. Clin Cancer Res 2008, 14, (10), 3036-3043.


Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


44.

Ray, S.; Chen, Z.; Pullambhatla, M.; Mease, R. C.; Pomper, M. G.

A preclinical

comparative study of Ga-68-labeled DOTA-, NOTA-, and HBED-CC-chelated PSMA-targeted radiotracers. J Nucl Med 2015, 56. 45.

Boswell, C. A.; Sun, X.; Niu, W.; Weisman, G. R.; Wong, E. H.; Rheingold, A. L.;

Anderson, C. J.

Comparative in vivo stability of copper-64-labeled cross-bridged and

conventional tetraazamacrocyclic complexes. Journal of medicinal chemistry 2004, 47, (6), 1465-74. 46.

Kinoshita, Y.; Kuratsukuri, K.; Landas, S.; Imaida, K.; Rovito, P. M., Jr.; Wang, C. Y.;

Haas, G. P. Expression of prostate-specific membrane antigen in normal and malignant human tissues. World journal of surgery 2006, 30, (4), 628-36. 47.

Banerjee, S. R.; Pullambhatla, M.; Foss, C. A.; Nimmagadda, S.; Ferdani, R.; Anderson,

C. J.; Mease, R. C.; Pomper, M. G. (6)(4)Cu-labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer. Journal of medicinal chemistry 2014, 57, (6), 265769. 48.

Villaraza, A. J.; Bumb, A.; Brechbiel, M. W.

Macromolecules, dendrimers, and

nanomaterials in magnetic resonance imaging: the interplay between size, function, and pharmacokinetics. Chemical reviews 2010, 110, (5), 2921-59. 49.

Kobayashi, H.; Sato, N.; Hiraga, A.; Saga, T.; Nakamoto, Y.; Ueda, H.; Konishi, J.;

Togashi, K.; Brechbiel, M. W. 3D-micro-MR angiography of mice using macromolecular MR contrast agents with polyamidoamine dendrimer core with reference to their pharmacokinetic properties. Magnet Reson Med 2001, 45, (3), 454-460. 50.

Lesniak, W. G.; Mishra, M. K.; Jyoti, A.; Balakrishnan, B.; Zhang, F.; Nance, E.;

Romero, R.; Kannan, S.; Kannan, R. M. Biodistribution of Fluorescently Labeled PAMAM



Page 36 of 50

Dendrimers in Neonatal Rabbits: Effect of Neuroinflammation. Mol Pharmaceut 2013, 10, (12), 4560-4571. 51.

Silver, D. A.; Pellicer, I.; Fair, W. R.; Heston, W. D.; Cordon-Cardo, C. Prostate-specific

membrane antigen expression in normal and malignant human tissues. Clin Cancer Res 1997, 3, (1), 81-5. 52.

Barinka, C.; Rojas, C.; Slusher, B.; Pomper, M.

Glutamate carboxypeptidase II in

diagnosis and treatment of neurologic disorders and prostate cancer. Current medicinal chemistry 2012, 19, (6), 856-70. 53.

Ma, D.; Hopf, C. E.; Malewicz, A. D.; Donovan, G. P.; Senter, P. D.; Goeckeler, W. F.;

Maddon, P. J.; Olson, W. C. Potent antitumor activity of an auristatin-conjugated, fully human monoclonal antibody to prostate-specific membrane antigen. Clin Cancer Res 2006, 12, (8), 2591-6. 54.

Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.;

Blattler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.; Wong, W. L.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer research 2008, 68, (22), 9280-90. 55.

Ray Banerejee, S.; Kumar, V.; Lisok, A.; Plyku, D.; Novakova, Z.; Wharram, B.;

Brummet, M.; Barinka, C.; Hobbs, R. F.; Pomper, M. G. Evaluation of

111In-DOTA-5D3,

a

surrogate spect imaging agent for radioimmunotherapy of prostate-specific membrane antigen. J Nucl Med2019 60, 400-06


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Tables

Table 1. Characteristics of G4(MP-KEU) and G4(Ctrl). D Rh MCC

MP-KEU

Bdiol MW (Da)

Size (nm)

ZP (mV)

G4(Ctrl)

2

3

0

0

37

23219

6

-0.3

G4(MP-KEU)

2

5

22

10

102

27360

5

-1.3

D - DOTA, Rh - rhodamine, MCC - linker, MP-KEU - PSMA targeting moiety, Bdiol - butane1,2-diol, MW - molecular weight size - number weight hydrodynamic size, resolution of used Malvern zetasizer is 2 nm and subtle difference in the size distribution observed for G4(Ctrl) and G4(M-PKEU) (see Figure 1F and S1B) is within experimental error, ZP - zeta potential.



FIGURES

Scheme 1. Synthesis of G4(MP-KEU) and G4(Ctrl). A - Synthesis of MP-KEU (MP- 5mercapto pentanamide, K - lysine, E - glutamic acid, U - urea) PSMA targeting moiety; B and C - schemes illustrating surface modifications of PAMAM G4(NH2)64 dendrimer leading to formation of G4(MP-KEU) PSMA targeted and G4(Ctrl) control dendrimers. Average number of conjugated functionalities was calculated based on increase of molecular weight upon each synthetic step, as detected by MALDI-TOF mass spectrometry.


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Figure 1. Purification and characterization of MP-KEU and G4(MP-KEU). A and B - RPHPLC and ESI-MS of MP-MP-KEU, demonstrating high purity of the PSMA-targeting moiety; 39 ACS Paragon Plus Environment


C - RP-HPLC purification of G4(MP-KEU), which was collected between 10 and 13 min of elution; D - RP-HPLC profile of G4(MP-KEU) with the UV-Vis spectrum recorded under the peak, indicating covalent attachment of rhodamine to the nanoparticles; E - MALDI-TOF spectra, illustrating increase of the molecular weight upon each modification step of dendrimer’s terminal primary amines (G4(NH2)64 - generation 4 amine terminated PAMAM dendrimer used as starting material and consecutive conjugates obtained through conjugation of 2 DOTA molecules (2), 3 rhodamines (3), 22 MCC heterobifunctional linkers (4), 10 MP-KEU targeting moieties (5) and capping of primary amines with butane-1,2-diol moieties (6), G4(MP-KEU) final PSMA targeted dendrimer); F - DLS of G4(MP-KEU), demonstrating narrow size distribution of the nanoparticles with the highest signal intensity between 4 and 6 nm averaging around 5 nm.


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Figure 2. In vitro evaluation of G4(MP-KEU). A - G4(MP-KEU) binding to PSMA+ PC3 PIP and PSMA- PC3 flu, 1×106 of each cell type was incubated with varied concentration of G4(MPKEU), CI - confidence interval; B - G4(MP-KEU) binding to PSMA+ PC3 PIP in the absence and presence of 1 mM of ZJ-43, incubation was carried out using 5×105 cells; C - competitive 41 ACS Paragon Plus Environment


binding assay of G4(MP-KEU) to PSMA+ PC3 PIP cells against ZJ-43, 7×106 cells were incubated with 1 µM of G4(MP-KEU) and increasing concentration of ZJ-43 ranging from 1 pM to 1 mM; D - summary of G4(MP-KEU) in vitro binding to PSMA+ PC3 PIP and PSMA- PC3 flu cell lines, **** P < 0.001; E - Epi-fluorescence microscopy of PSMA+ PC3 PIP, PSMA- PC3 flu cells after incubation with 150 nM of G4(MP-KEU) or 150 nM of G4(MP-KEU) plus 10 µM of ZJ-43 for 2 h at 37oC, scale bar: 50 µm. All panels demonstrate in vitro PSMA specificity of G4(MP-KEU).


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Figure 3. Ex vivo biodistribution of G4(MP-KEU) and G4(Ctrl). Representative optical images of organs and tumors from male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA43 ACS Paragon Plus Environment


PC3 flu xenografts harvested 24 h post IV injection of: A - G4(MP-KEU) (75 µg, 2.75 nmol, n=4 ), B - G4(MP-KEU) (75 µg, 2.75 nm, n=4) plus ZJ-43 (1 mg, 3.29 µmol, n=4), C - G4(Ctrl) (64 µg, 2.76 nmol, n=4) and D - saline (n=2). Images were acquired on a Xenogen IVIS Spectrum optical imaging system with excitation at 535 nm and emission at 580 nm. E fluorescence images showing differential uptake of G4(MP-KEU) in PSMA+ PC3 PIP and PSMA- PC3 flu tumors; F - semi-quantitative analysis of G4(MP-KEU) accumulation in PSMA+ PC3 PIP and PSMA- PC3 flu tumors, **** P < 0.001; G-I - epifluorescence microscopy illustrating distribution of G4(MP-KEU) in PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and kidney acquired using freshly cut unstained sections; I-M - epifluorescence microscopic images, illustrating PSMA expression and co-localization with G4(MP-KEU) nanoparticles in PSMA+ PC3 PIP tumor (arrows).


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Figure 4. Radiolabeling of G4(MP-KEU) and in vitro evaluation of [64Cu]G4(MP-KEU). A Radio-HPLC chromatogram of the unpurified [64Cu]G4(MP-KEU) demonstrating 80.6 % radiolabeling efficiently; B - Radio-HPCL profile of the [64Cu]G4(MP-KEU) obtained after ultrafiltration, showing high radiochemical purity of the radiotracer; C - in vitro binding of [64Cu]G4(MP-KEU) to PSMA+ PC3 PIP and PSMA- PC3 flu cell lines and blocking with 1 µM of ZJ-43 indicating PSMA specificity of nanoparticles, **** P < 0.001. With highlighted corrections



Figure 5. In vivo evaluation of [64Cu]G4(MP-KEU). A - Decay corrected volume-rendered PET-CT images of male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu xenografts injected with 9.25 MBq of [64Cu]G4(MP-KEU) (upper panel, n=3) or 9.25 MBq of [64Cu]G4(MP-KEU) with 50 mg/kg of ZJ-43 (lower panel, n=1); B - Ex vivo biodistribution of [64Cu]G4(MP-KEU) at 3 h, 24 h and 48 h after injection, in the same tumor model (n=4 or 3), ** P < 0.02. Both PET-CT and biodistribution results indicate PSMA mediated [64Cu]G4(MP-KEU) uptake in PSMA+ PC3 PIP tumor.


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Figure 6. Pharmacokinetics of [64Cu]G4(MP-KEU) and [64Cu]G4(Ctrl).

Ex vivo

biodistribution of [64Cu]G4(MP-KEU) and [64Cu]G4(Ctrl) at 3 h, 24 h and 48 h after injection in 47 ACS Paragon Plus Environment


male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu xenografts (n=4). **** P < 0.001. Results indicate no preferential uptake of [64Cu]G4(Ctrl) in PSMA+ PC3 PIP vs. PSMAPC3 flu tumors, rapid renal clearance and minor hepatic accumulation.


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Figure 7. Evaluation of [111In]G4(MP-KEU) and [111In]G4(Ctrl). A - Ex vivo biodistribution of [111In]G4(MP-KEU) and [111In]G4(Ctrl) in male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu xenografts (n=4), *** P < 0.005; B - Decay corrected, volume-rendered SPECT-CT images of male NOD-SCID mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu xenografts injected with 29.6 MBq of [111In]G4(MP-KEU) or [111In]G4(Ctrl). Mice were imaged concurrently, side-by-side on the gantry. Ex vivo biodistribution and SPECT-CT results validate PSMA specificity of [111In]G4(MP-KEU).



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Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a

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